Plasmon resonant particles, methods and apparatus

ABSTRACT

A method and apparatus for interrogating a target having a plurality of plasmon resonant particles (PREs) distributed in the target are disclosed. In the method, a field containing the target is illuminated, and one or more spectral emission characteristics of the light-scattering particles in the field are detected. From this data, an image of positions and spectral characteristic values in the field is constructed, allowing PREs with a selected spectral signature to be discriminated from other light-scattering entities, to provide information about the field. Also disclosed are a novel PRE composition for use in practicing the method, and a variety of diagnostic applications of the method.

This application claims priority under 35 U.S.C. §120 to ProvisionalApplication Ser. No. 60/038,677, filed Feb. 20, 1997, entitled“Preparation and Use of Plasmon Resonant Particles”, which is herebyincorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates plasmon resonant entities (PREs), orparticles, to methods of interrogating a field containing PREs, and toapparatus for carrying out the method, and to various applications ofPREs.

BACKGROUND OF THE INVENTION

There are a number of important commercial and scientific applicationsof interrogating a target for information about the target. For example,the aim of analyte diagnostic tests and methods is to detect thepresence and/or amount of an analyte (the target). The target analytemay be detected by reacting the analyte with a detectable reporter that(i) can bind specifically to the analyte and (ii) is detectable withsuitable detecting tools. The reporter may, for example, be a colored orfluorescence molecule, or a colloidal metal, or a reporter such as aradiolabel that requires special film or scintillation equipment for itsdetection.

In some diagnostic applications, it is desirable to detect proximityrelationships in a target analyte, as evidenced by the interactionbetween two proximately located probes on the target analyte. This formsthe basis of so-called homogeneous assays, where the presence of ananalyte is determined by a detectable probe proximity effect observedwhen two distinct probes are brought together on closely spaced sites onthe analyte. As an example, two fluorescent molecules, when broughttogether, may exhibit a detectable fluorescence quenching or anon-radiative energy transfer effect that acts to shift the Stokesradius between the excitation and emission peaks.

A chemical, biochemical, or biological target may be interrogated by avariety of chemical and spectrographic methods to determine chemicalstructure, the presence of certain chemical groups, or the environmentof the chemical groups. Notable among these methods are magneticresonance methods for determining chemical structure and chemical groupenvironment, spectroscopic methods, such as UV, IR, Raman, ORD, and CDspectroscopy, for detecting specific chemical groups, and massspectroscopy for determining structure by fragment molecular weightanalysis.

Surface chemical analysis of a target sample may be carried out bybombarding the surface with high-energy particles, e.g., electrons, anddetecting the energy of atoms that are ejected from the surface.Electron Spectroscopy for Chemical Analysis (ESCA) is an example of suchan approach.

Often it is desirable to establish spatial and/or distance relationshipsin a target, generally requiring interrogation by microscopy. Lightmicroscopy has the advantage of simplicity, ease of sample preparation,and the feature that the sample can be examined in a “wet” condition.Its disadvantage is the relatively low resolving power, directly relatedto the wavelength of the illumination source (in the 400-650 nm range)and inversely proportional to the numerical aperture of the lens systems(at best, about 1.4), limiting resolution to several hundred nm).

High-energy beam microscopes, such as the transmission electronmicroscope (TEM) and the scanning electron microscope (SEM) can achieveresolution down to the low nm range, but require a high-vacuumenvironment of the target sample, limiting applications with biologicalsamples. Atomic force microscopy (AFM) is useful for interrogatingsurface features of a target sample, also with a resolution in the lownm range. The method is limited to surface effects.

Radiographic and scintigraphic methods for detecting and/or localizingsites of high-energy emission are also widely used. These methods tendto be quite sensitive, being able to detect very low numbers ofhigh-energy emission events, but suffer from relatively high-cost andpoor resolution when target spatial information is desired.

Despite the variety of methods currently available, there are a numberof target-interrogation tasks of commercial and scientific interest thatare difficult or impossible with current methods. Among these are:

1. Detecting single (or only a few) molecular events, such as thepresence of one or a few binding sites, or one or a few enzymic sites ona target. This capability would open up new diagnostic applications,e.g., related to the presence or absence of specific intracellularevents, and reduce the amount of sample material needed for a reliableassay and allow miniaturization of the assay.

2. Resolving sub-wavelength distance relationships in a biologicaltarget in a natural hydrated state. As noted above, subwavelengthresolution by high-energy beam microscopy requires the sample target tobe in a desiccated state, precluding the observation of natural cellularprocesses, including subwavelength movement of cellular components, andallows the user to perturb the sample during observation.

3. Direct spatial mapping of selected target sites on a biologicaltarget, such as direct mapping of selected sequences in a chromosome forpurposes of chromosome mapping. Currently, this type of information iseither not practical, or in the case of chromosome mapping, is notpossible at high resolution and precise localization of gene sequences.

4. Optical reading of microencoded information. The ability to detectunique patterns of individual reporter groups would have importantapplications in forensics, information storage, metrology, and securityidentification microcodes.

It would therefore be desirable to provide a method and apparatus forinterrogating a field for the type of information outlined above that isimpractical or impossible to obtain by prior art methods.

It would also be desirable to apply the method to various diagnosticsapplications, to achieve improved sensitivity, spatial and distanceinformation, ease of sample preparation, and flexibility in the type oftarget sample that can be interrogated.

SUMMARY OF THE INVENTION

In one aspect, the invention includes a method of interrogating a fieldhaving a plurality of PREs distributed therein. The method includes thesteps of illuminating the field with an optical light source, anddetecting a spectral emission characteristic for individual PREs andother light scattering entities in the field. From this information isconstructed a computer image of the positions and values of the emissionspectral characteristic of individual PREs and other light-scatteringentities present in the field, as a basis for discriminating PREs with aselected spectral signature from other light-scattering entities in thefield, to provide information about the field.

The illuminating step may be carried out at different frequency bands,where the spectral emission characteristic of individual PREs and otherlight scattering entities in the field are detected at each such band.

Alternatively, the illuminating step may include exposing the field to aplurality of narrowband pulses of light which vary in duration, todetect variations in emitted light intensity produced by variations induration.

In another embodiment, where the field preferably includes at least somenon-spherical PREs, the illuminating step may involve exposing the fieldto polarized light at different orientations and/or different angles ofincidence. The detecting step includes detecting a change in value of aspectral emission characteristic as a function of incident lightpolarization orientation or angle of incidence.

The detecting step may include simultaneously detecting the values of aspectral emission characteristic of individual PREs and other lightscattering entities in the field at a plurality of defined spectralbands. Alternatively, the spectral emission characteristic values ofindividual PREs and other light scattering entities in the field may bedetected sequentially at a plurality of defined spectral bands.

The PREs may be formed in or added to the field by metal enhancingnucleation centers in the field, by adding pre-formed PREs to a targetin the field, or by making PREs at target sites in the field, e.g., byphotolithographic methods.

The method may be practiced to discriminate PREs with a selectedspectral signature from all other light-scattering entities in thefield. The spectral emission characteristic that is detected, as a basisfor the discrimination, is typically peak position, peak intensity, orpeak width at half intensity of the spectral emission curve, peakhalfwidth in the image plane, and/or polarization or angle of incidenceresponse. Other emission spectral characteristics, such as response topulsed beam illumination, are also contemplated.

The same spectral characteristics, either alone or in combination, areuseful for discriminating (i) PREs from non-PRE light-scatteringentities, (ii) one selected type of PRE from another, and (iii) PREsthat are interacting through proximity effects from non-interacting PREs(typically PRPs).

In another embodiment, the PREs have a surface localized fluorescent orRaman-active molecular entities, e.g., Raman-active molecules, and thedetecting includes detecting plasmon-resonance induced fluorescenceemission or Raman spectroscopy emission from one or more of saidentities.

The method may be carried out to yield information about (i) the totalnumber of PREs of a selected type in a field, (ii) the spatial patternof PREs having a selected range of values of a selected spectralcharacteristic in the field, (iii) a distance measurement between twoadjacent PREs, particularly PREs separated by a distance less than theRayleigh distance, (iv) a change in the environment of the field, e.g.,dielectric constant, that affects the value of a plasmon resonancecharacteristics, or (v) motion of PREs in the field.

In another aspect, the invention includes apparatus for interrogating afield having a plurality of PREs distributed therein, for example, inpracticing the above method for interrogating a field. The apparatusincludes an optical light source for illuminating the field, and anoptical detector for detecting values of a spectral emissioncharacteristic of individual PREs and other light scattering entities inthe field, when the field is illuminated by the light source.

Also included in the apparatus is an image processor operativelyconnected to the detector for constructing, from signals received fromthe detector, a computer image of the positions and detected values ofthe emission spectral characteristic of individual PREs and such otherlight-scattering entities present in the field, and a discriminator fordiscriminating PREs with a selected spectral signature from otherlight-scattering entities in the computer image, i.e., a selected rangeof values of a selected spectral emission characteristic. The apparatusis constructed to display (or store) information about the field basedon the information about the selected PREs.

One preferred light source is a bright field/dark field lens fordirecting light onto the field. The illumination source mayalternatively be a bright field lens, a dark field lens, a polarizer forproducing polarized-light illumination source, such as a plane-polarizedlight source, a TIR, a pulsed beam, an epi-illumination system in whichlight is reflected by a half-silvered mirror through a dark field/brightfield lens, and a dark field condenser lens. The light source mayinclude means for separately with field with light having differentexcitation wavelengths.

The optical detector may include structure for spectrally separatinglight emitted from the PREs. The detector in this embodiment operates toform a computer image of the positions and emission spectralcharacteristic values of individual PREs and such other light-scatteringentities present in the field at each of a plurality of differentemission wavelengths.

The optical detector may include a two dimensional array of opticalfibers, a grating or prism for responding to the output of the opticalfibers when aligned to act as a line source of light from the array, anda two-dimensional detector array for responding to the spread-outspectral light from each fiber in the line source of light.

The image processor may operate to construct an image of field positionsand associated values of peak position, peak intensity, or peak width athalf intensity of the spectral emission curve, peak halfwidth in theimage plane, and/or polarization or angle of incidence response.

In other embodiments, where the PREs have surface associated fluorescentor Raman-active molecular entities, the image processor operates toconstruct an image of field positions and fluorescence peak ofplasmon-resonance induced fluorescence, or a Raman spectral feature inplasmon-resonance induced Raman spectral emission.

The discriminator may operate to discriminate a selected type of PREfrom all other light-scattering entities in the field, PREs from non-PREsubwavelength light-scattering particles, including: (i) PREs fromnon-PRE light-scattering entities, (ii) one selected type of PRE fromanother, and (iii) PREs that are interacting through proximity effectsfrom non-interacting PREs (typically PRPs).

The information displayed by the apparatus may be related to informationabout (i) the total number of PREs of a selected type in a field, (ii)the spatial pattern of PREs having a selected spectral characteristic inthe field, (iii) a distance measurement between two adjacent PREs,particularly PREs separated by a distance less than the Rayleighdistance, (iv) a change in the environment of the field, e.g.,dielectric constant, that affects a plasmon resonance characteristics,or (v) motion of PREs in the field.

In another aspect, the invention includes a composition of plasmonresonant particles (PRPs) having one or more populations of PRPs. Thecomposition is characterized by: (a) the PRPs have a width at halfheightof less than 100 nm; (b) the PRPs in a single population are all within40 nm of a defined wavelength; (c) at least 80% of the PRPs in thecomposition satisfying criterion (a) are in one or more of thepopulations and have a spectral emission wavelength in a singlerange >700 nm, 400-700 nm, or <400 nm; and (d) each population has atmost a 30% overlap in number of PRPs with any other population in thecomposition. The composition may be used in practicing the abovetarget-interrogation method, and/or in conjunction with the abovetarget-interrogation apparatus.

In one embodiment at least 80% of the PRPs in the composition are in oneor more of the populations and have a spectral emission wavelength inthe 400-700 nm wavelength range. Also in this embodiment, the particleshave a composition formed of a solid silver particle, a silver particlewith a gold core, or a particle with a dielectric core and an outersilver shell of at least about 5 nm.

In one general embodiment, for use particularly in a variety ofdiagnostic applications, the particles have localized at their surfaces,(i) surface-attached ligands adapted to bind to ligand-binding sites ona target, where the ligand/ligand-binding sites are conjugate bindingpairs, (ii) fluorescent molecules, (iii) Raman-active molecularentities, and (iv) a blocking reagent to prevent non-specific binding,(v) a coating with functional groups for covalent coupling to the PRPs,or (vi) combinations of (i)-(v).

The localized ligand may be one of a conjugate pair, such asantigen/antibody, hormone/receptor, drug/receptor, effector/receptor,enzyme/substrate, lipid/lipid binding agent and complementary nucleicacids strands.

The composition may have first and second populations of PRPs havingfirst and second different surface localized molecules or entities. Foruse in identifying a target having first and second ligand-bindingsites, the first and second surface bound molecules are first and secondligands effective to bind to the first and second ligand-binding sites,respectively. As an example, the first and second surface-localizedmolecules are oligonucleotides having sequences that are complementaryto first and second proximate sequence regions of a targetpolynucleotide. As another example, the first and secondsurface-localized entities may be Raman-active molecular entities withdifferent Raman spectral characteristics.

The composition may contain first and second populations of PRPs, eachwith a different shape, at least one of which is spherical ortetrahedral.

In still another aspect, the invention includes a diagnostic method foruse in detecting the presence of, or information about, a target havinga molecular feature of interest. The method includes contacting thetarget with one or more PREs (preferably PRPs) having surface localizedmolecules, to produce an interaction between the molecular feature andthe localized molecules, illuminating the target with an optical lightsource, and determining the presence of or information about the targetby observing a plasmon resonance spectral emission characteristic of oneor more PRPs after such interaction with the target. The diagnosticmethods may be carried out, for example, by the abovetarget-interrogation method above, using the above target-interrogationapparatus.

In a general embodiment, the target contains a ligand-binding site, andthe surface-localized molecule is a ligand capable of forming aligand/ligand-binding complex with the target. The binding interactionis detected by detecting a plasmon resonance spectral emissioncharacteristic of the complex. The surface localized ligand may be, forexample, a polynucleotide, oligonucleotide, antigen, antibody, receptor,hormone, enzyme, or drug compound.

In a solid-phase format of the method, the target is washed to removePRPs not bound to the target through a ligand/ligand-bindinginteraction, before detecting complex.

In a homogeneous phase of the method, the interaction of the PRE(s) withthe target is effective to produce either a plasmon-resonance spectralemission characteristic which is distinguishable from that of thenon-interacting PREs, or separate diffraction centers, where the twoPREs have different peak wavelengths. By detecting one of thesefeatures, the presence of the diagnostic interaction can be determined.

In one homogeneous-phase embodiment, the PRE(s) containsurface-localized fluorescent reporter molecules, and the interaction ofa PRE with the target or with another PRE at the target is effective todetectably alter a plasmon-resonance induced spectral emissioncharacteristic of the fluorescent molecules on the PRE.

In another embodiment, the PRE(s) contain surface-localized Raman-activemolecular entities, and the interaction of a PRE with the target or withanother PRE at the target is effective to detectably alter aplasmon-resonance induced spectral emission characteristic of theRaman-active molecular entities on the PRE.

In still another embodiment, the target has two or more proximatelyspaced ligand-binding sites, and the complex that forms includes atleast two proximately spaced PREs that have a spectral emissionsignature different from that of PREs in the absence of binding to thetarget, e.g., a change in the spectral emission curve of the complex,where the two PREs have substantially the same peak wavelength.Alternatively, where the two PREs have different peak wavelengths, theindividual PREs may be interrogated at the two different wavelengths,and the distance between PREs determined by the distance between centersof the two diffraction patterns in the image plane. The embodiment maybe practiced, for example, by reacting the target with first and secondpopulations of PREs having surface-localized first and second ligands,respectively, for binding to the first and second ligand binding sites,respectively.

For use in forming a spatial image of the target, where the target hasmultiple ligand-binding sites, contacting the PREs with the targetproduces binding at multiple sites. The detecting step includesconstructing a spatial image of the sites of PRE attachment to thetarget, which is indicative of the relative spacings of theligand-binding sites in the target.

One application involves the mapping of closely spaced regions in apolynucleotide, where the detecting includes observing the spacingbetween centers of the diffraction patterns of the PREs in the imageplane of the PREs.

Another application involves gene mapping, e.g., by binding PREs withdifferent complementary surface-localized oligonucleotides to a targetpolynucleotide, with such in an extended condition.

In another embodiment, for use in detecting target sequence mutations orfor sequencing by hybridization, the target is an array ofdifferent-sequence oligo- or polynucleotides. The array is contactedwith one or more PREs having one or more surface-localized testpolynucleotides, under conditions which allow the PRE'ssurface-localized polynucleotides to hybridize with the target arrayoligo- or polynucleotides. After washing the target to remove unboundPREs, a spectral emission characteristic of PREs at each region of thearray is detected, to determine the pattern of polynucleotide binding tothe array.

In another embodiment, the target is a polynucleotide present as aseparated band in an electrophoresis gel, and the contacting is carriedout by exposing the surface of the gel to PREs under hybridizationconditions. This method simplifies the Southern hybridization method byeliminating a DNA band transfer step.

In another general embodiment of the method, the molecular feature ofinterest is a molecule which functions catalytically to break a bondbetween two atoms in a molecular chain. The PRE reagent in the method isa pair of PREs linked by said chain, where the linked PREs may have aspectral emission spectrum different from that of the individual, i.e.,separated, PREs. The contacting is carried out under conditionseffective to cleave the molecular chain. The presence of the cleavingagent is detected by the disappearance of the linked-PRE spectralemission signature, or the appearance of the individual-PRE spectralemission characteristic, or a change in the detected distance betweenthe two PREs.

In another aspect, the invention includes a composition of plasmonresonant particles (PRPs) characterized by: (a) the PRPs have a width athalfheight of less than 100 nm; (b) at least 80% of the PRPs in thecomposition satisfying criterion (a) are in one or more of thepopulations and have a spectral emission wavelength in a singlerange >700 nm, 400-700 nm, or <400 nm; and (c) surface localized ligandsadapted to bind to ligand-binding sites on a target, where theligand/ligand-binding sites are conjugate binding pairs, (ii)fluorescent molecules, or (iii) Raman-active molecular entities.

The invention further includes a variety of PRE compositions and methodsdiscussed in Section VI of the Detailed Description of the Invention.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the relative scattering intensity of two opticallyobservable plasmon resonant entities with disparate peak scatteringwavelengths.

FIG. 2 is a graph of the relative scattering intensity of four opticallyobservable plasmon resonant entities with similar peak scatteringwavelengths.

FIG. 3 is a schematic illustration of one embodiment of a darkfieldmicroscope detection system suitable for the observation of plasmonresonant entities.

FIG. 4 is an illustration of a liquid analog to a solid-immersion-lenswhich may be used to observe plasmon resonant entities.

FIG. 5 is an illustration of a total internal reflection type samplestage suitable for use in the observation of plasmon resonant entities.

FIG. 6 illustrates a reflecting brightfield/darkfield lens suitable forPRE imaging.

FIG. 7 is a reproduction of a transmission electron microscope image oftwo plasmon resonant particles.

FIG. 8 is a graph of light intensity as a function of position in theimage plane at two different bandwidths emitted by the plasmon resonantparticles shown in FIG. 7.

FIG. 9 is a graph showing the results of an assay performed with plasmonresonant labels.

FIG. 10A illustrates a focused light beam having intensity profilecharacteristics measurable with plasmon resonant entities

FIG. 10B illustrated the placement of a plasmon resonant entity withinthe focused light beam of FIG. 10A.

FIG. 11 is a Raman signature from a Raman-active PRE.

FIG. 12 is a chicken skeletal muscle section whose ryanodine receptorshave been labeled with anti-ryanodine PRPs.

FIG. 13 is a Drosophila polytene chromosomes where a specific gene hasbeen labeled by PRPs.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Definitions

The following terms have the definitions given below, unless indicatedotherwise:

“Plasmon resonant particle” or “PRP” denotes a single piece or fragmentof material, e.g., spherical particle, which elicits plasmon resonancewhen excited with electromagnetic energy. A plasmon resonant particlecan be “optically observable” when it exhibits significant scatteringintensity in the optical region, which includes wavelengths fromapproximately 180 nanometers (nm) to several microns. A plasmon resonantparticle can be “visually observable” when it exhibits significantscattering intensity in the wavelength band from approximately 400 nm to700 nm which is detectable by the human eye. Plasmon resonance iscreated via the interaction of incident light with basically freeconduction electrons. The particles or entities have dimensions, e.g.,diameters preferably about 25 to 150 nm, more preferably, about 40 to100 nm.

The term “plasmon resonant entity” or “PRE” is used herein to refer toany independent structure exhibiting plasmon resonance characteristic ofthe structure, including (but not limited to) both plasmon resonantparticles (PRPs) and combinations or associations of plasmon resonantparticles as defined and described above. A PRE may include either asingle PRP or an aggregate of two or more PRPs which manifest a plasmonresonance characteristic when excited with electromagnetic energy.

A “field having a plurality of PREs distributed therein” is a one-,two-, or three-dimensional region, for example, a target or portion orregion of a target having PREs attached or otherwise distributedtherein, such that the PREs in the field, when illuminated with anoptical light source, exhibit plasmon resonance.

A “spectral emission characteristic” refers to a spectral scatteringcharacteristic of a PRE related to the plasmon resonance of the PRE, asdiscussed in Section III. As used herein, “emission”, as applied toPREs, means scattered light produced or excited by plasmon resonance.

The “value” of a spectral emission characteristic is the qualitative orquantitative value of the emission feature, e.g., the value of thedetected peak intensity, peak wavelength, or peak width at half maximum.

A “selected spectral signature” refers to a selected range of values ofa selected spectral emission characteristic, e.g., a range of spectralpeak intensity values.

A “computer image of the positions and values of the emission spectralcharacteristic” refers to a matrix which associates each region in afield being interrogated with one or more spectral emissioncharacteristic values or signature measured for a light-scatteringentity in that region. The image may be a matrix of stored values, ormay be an actual image showing the locations of light-scatteringentities in one dimension or plane, e.g., the x-y plane, and theassociated spectral emission value in another dimension, e.g., thez-axis.

A “ligand” is a chemical species, typically a biochemical species, thatis capable of forming a specific, typically high-affinity bond with a“ligand-binding” site or molecule. The ligand/anti-ligand form aconjugate pair that can include, for example, antigen/antibody,hormone/receptor, drug/receptor, effector/receptor, enzyme/substrate,lipid/lipid binding agent and complementary nucleic acids strands.

A “Raman-active molecular entity” is a molecule, molecular complex, orparticle, e.g., silicon particle, that displays a Raman spectroscopicsignature, preferably through resonance Raman excitation, when excitedby electric fields of a plasmon-resonating particle to which themolecular entity is attached.

“Surface-localized” ligands and other species refer to molecular speciesthat are attached to a PRE by covalent or other molecular forces, e.g.,electrostatic or dispersion forces, or which are embedded in a shell orother surface coating on a PRE.

II. Plasmon Resonance

The present invention utilizes one or more of a number of spectralemission characteristics of conductive plasmon-resonance particles (PRPsor PREs) to interrogate a field for a variety of types of information,including the presence or absence of a target, spatial features of atarget, the environment of a target, number and/or spatial distributionof a selected type of target binding sites, and distance relationshipsin the target, as will be detailed in Sections III-VI below.

Plasmon resonant entities (PREs) or plasmon resonant particles (PRPs)scatter incident light, and the resulting scattered light has afrequency spectrum characteristic of the particle. A general theorydescribing the interaction of an incident electromagnetic wave with aspherical particle which successfully predicts this resonant scatteringwas developed early in the 20th century (H. C. Van Ve Hulst, LightScattering by Small Particles, Wyley, N.Y., 1957). In a metallic sphere,the incident electromagnetic field induces oscillations, referred to as“plasmons”, in the nearly free conduction electrons of the metal, andthese plasmons produce an emitted electromagnetic field. For somematerials, and for the optimum choice of particle size, shape, andmorphology, there will be a maximum scattering efficiency at awavelength characteristic of the scattering particle and its surroundingmedium. For some materials, the intensity of the emitted light issufficient for observation under an optical microscope. Silver particlesare the most notable exhibitors of this effect, as the wavelength of theresonantly scattered light can be in the visible region of the spectrum.

Theoretical calculations correctly predict that the resonantly scatteredwavelength of a spherical particle will increase, or be “red-shifted”,with increasing particle diameter and with increasing dielectricconstant of the surrounding material. For spherical particles, dipoleresonance produces a scattered frequency spectrum having a single peakat a wavelength which is dependent on the material the particle is madefrom the size of the particle, the shape of the particle, the morphologyof the particle, and the local environment. Larger particles have alonger dipole scattering peak wavelength, and smaller particles have ashorter dipole scattering peak wavelength. The spectrum of scatteredlight may also contain contributions from a particle's quadrupoleresonance. For a given shape, a resonant particle scatters predominantlyin a particular wavelength band depending on the composition and size ofthe particle.

The conductive portion responsible for the plasmons can take manydifferent forms, including solid geometric shapes such as spheres,triangular parallelpipeds, ellipsoids, tetrahedrons, and the like, ormay comprise spherical, cylindrical, or other shape shells. It is alsotrue that a dielectric sphere of similar dimensions, having silver orgold on its surface will also exhibit plasmon resonances, assuming theshell has a thickness of at least about 3 nm, preferably 5 nm or more.

It can further be appreciated that contact or near contact between twoplasmon resonant particles will produce an electromagnetic couplingbetween the particles, thereby producing an entity with properties insome ways similar to a single particle having a size equal to the sum ofthe two particles in contact. Aggregations of many plasmon resonantparticles can therefore also exhibit plasmon resonance withcharacteristics dependent on the geometry and nature of theconglomerate.

Another feature of plasmon creation in a metallic particle is thegeneration of enhanced electric fields in the region near its surface.Interactions between this electric field and nearby materials cansignificantly alter both the scattering characteristics of the resonantparticle and the nearby material. For example, Surface Enhanced RamanSpectroscopy (SERS) exploits the localized plasmon resonance inroughened or particle coated silver films to enhance the Ramanscattering of various materials by as much as six orders of magnitude.In this technique, Raman scattering from the materials of interest isobserved, and the local field generated by the plasmons is used toenhance the intensity of that scattering.

Referring now to FIG. 1, a graph of the relative scattering intensity oftwo PRPs is illustrated, demonstrating that different PREs can havedifferences in spectral characteristics that are easily detected.Although the spectra shown in FIG. 1 could be produced by eitherindividual PRPs or PREs of a more complex structure, it will be assumedthat the source of the scattered light spectra illustrated in FIG. 1 isfrom PRPs for explanatory purposes.

In FIG. 1, the relative intensity of scattered light in arbitrary unitsis plotted against wavelength in nanometers. The individual spectra oftwo different PRPs are shown—one, spectrum 3, having a peak emission 5at approximately 460 nm, and a second, spectrum 7, having a peakemission 9 at approximately 560 nm. In this figure, the light intensityof the light emitted by each of the two PREs were individuallynormalized to 1.0. The shape of each spectrum is approximatelyLorentzian, with a width at half maximum of approximately 30 nm for theparticle with 470 nm peak, and approximately 50 nm for the particle with560 nm peak. As has been mentioned above, the light emitted byindividual PRPs can be visually observed with an appropriate opticalmicroscope. If the two PRPs with emission spectra illustrated in FIG. 1were so observed, the PRP with peak 5 at 470 nm would appear blue, andthe PRP with peak 9 at 560 nm would appear yellow.

FIG. 2 shows the spectral emission curves for a population of fourdifferent populations of PREs, each having an approximately homogeneousproperties. The spectra 10, 11, 12, 13 of the four PREs shown in thisfigure have peak emission wavelengths which vary from approximately 460nm to 480 nm. Visually, each of the four PREs which produce the spectrashown in FIG. 2 would appear blue in color. The four particles can bedistinguished, however, on the basis of spectral peak intensity, i.e.,peak height, or on the basis of the different spectral emission curves,for example, by comparing the ratios of peak height to peak width athalf peak height. Other spectral emission characteristics are discussedbelow.

III. Method and Apparatus for Interrogating a Field

In one aspect, the invention is directed to a method and apparatus forinterrogating a field having a plurality of PREs distributed therein.The method has three parts, in essence: (i) generating data about one ormore spectral emission characteristic(s) of PREs in the field, (ii) fromthis data, constructing a computer image of the PRE positions (regionsin a field) and values of the emission spectral characteristic ofindividual PREs and other light-scattering entities present in thefield, and (iii) by discriminating PREs with selected spectralcharacteristics in the image from other light-scattering particles inthe field, providing information about the field, e.g., a target in thefield.

A. Spectral Emission Characteristics

The invention contemplates detecting one or more of several types ofspectral emission characteristics, for generating an image oflight-scattering particles in the field. The spectral emissioncharacteristics of interest may be plasmon-resonance spectral featuresof a single PRP, a shift in spectral emission feature due to theinteraction of two or more PRPs in close proximity, or a fluorescent orRaman spectroscopic feature induced by the enhanced local electricfields interacting with fluorescent, luminescent, or Raman moleculeslocalized on PREs. The most important of characteristics, and the typeof information available from each, are the following.

Peak wavelength is the wavelength of the peak of the spectral emissioncurve, that is, the wavelength at which maximum intensity occurs. Peakwavelengths for the two spectral emission curves shown in FIG. 1 areindicated at 5 and 9, corresponding to wavelength values of 470 nm and560 nm, as described above.

The peak wavelength value can be determined in one a number of differentways, seven of which are described here. The implementation of each ofthe methods will be understood from the disclosed method, and for someof the methods, as discussed below in the description of the lightsource and detector in the apparatus of the invention. All of thesemethods are applicable to measuring the spectral curves for a pluralitysimultaneously. It will be appreciated that some of the methods are alsoapplicable to measuring the spectral curve of each light-scatteringentity in the field individually, for example, by rastering aphotodetector element over the plane of the field.

(i) The field is illuminated over a range of illuminating wavelengths,for example, at each of a series of narrowband illumination windowsthrough the visible light spectrum. Typically, a filter wheel interposedbetween a white light source and the field is employed to generate thenarrowband illumination frequencies.

(ii) Light emitted from the field is directed through a dispersiveelement, such as a prism, for breaking the emitted light into severalnarrowband frequencies, which are then each directed to a separatedetector array. As an example, a prism is used to break the emittedlight into red, green and blue components, each directed onto a separateCCD array.

(iii) Take the emitted field image into a dense bundle of opticalfibers, through a lens that, for example, magnifies eachlight-scattering spot corresponding to a PRE, such that its image fitsentirely in the core diameter of an optical fiber. Each fiber is thenbroken up by a dispersion element into spread out spectrum line ofdifferent frequencies, which is then read by a line of detector elementsin a two dimensional array. Thus each line in the field is read by a2-dimensional array, one array dimension corresponding to the spectralintensity at each of a plurality of frequencies, and the otherdimension, to different positions along an axis in the field. Thisapproach allows for simultaneous reading of a plurality of PREs at eachof a plurality of spectral wavelengths.

(iv) Illuminate with multiple narrow band light sources, e.g., 3 or 4separate laser lines in the red, green, yellow and blue. Each laser ischopped at a different frequency, typically all under 100 Hz. Theemitted light from the field is detected in a CCD that can be read at100 frames/sec. Computer analysis involving standard techniques is thenused to determine the amount of light of each color impinging on eachpixel in the CCD array, thereby allowing the spectral emission curve tobe constructed.

(v) The same information may be obtained by routing the scattered lightthrough an interferometer, as described for example, in U.S. Pat. No.5,539,517.

(vi) It is also a property of plasmon resonant particles that thescattered light undergoes a 180 degree phase shift relative to theincident light as the wavelength of incident light is swept through theresonant peak. At the peak wavelength, the phase difference is 90degrees. This phase shift can be detected, and the peak scatteringwavelength can be determined as that incident wavelength when a phaseshift of 90 degrees is observed.

(vii) The intensity of PRE light emission at a plurality of definedbandwidths can also be determined by exposing the PREs to short pulsesof incident light of varying duration. In particular, it is effective touse pulses approximating a step function increase or decrease, that is,with fast rise time or decay time of only 1 or 2 femtoseconds. Thescattering response of a PRE is that of a forced and damped oscillator,and near the resonant wavelength, the response of a PRE to narrowbandexcitation increases as the excitation pulse length increases. Away fromthe resonant wavelength, the response to narrowband excitation is small,and relatively independent of the excitation pulse length. Exposing aPRE to pulses of varying duration, but all advantageously less thanabout 500 femtoseconds, at a particular wavelength and noting how longit takes for the emitted energy to reach a steady state value providesinformation about how close that particular wavelength is to the PREresonant wavelength. By exciting the PREs to several series of durationvariable pulses, wherein each series has a different peak wavelength, acurve of scattering cross section versus wavelength can be generated.

The peak wavelength generally shifts toward the red (longer wavelengths)as the size of the PRE increases for silver and gold PREs. Peakwavelength values can also provided information about PRE shape. Shapechanges from spherical to hexagonal or triangular result predominantly ashift of peak wavelength toward the red. Dielectric-shell PRPs, i.e.,particles composed of an inner dielectric core encased in a conductivemetal also tend to have longer peak wavelengths than solid metalparticles of the same size.

Peak intensity is the intensity of the peak of the spectral emissioncurve, and may be expressed as an absolute or relative intensity value,as in FIG. 2, which shows four PREs with different relative peakintensities ranging from less than 3 to greater than 10. The peakintensity value is determined, as above, by one of a variety of methodsfor determining the spectral emission curves of the PREs, with intensitybeing determined at the peak wavelength.

The peak intensity will vary with material, morphology and shape. For aparticular PRE, the intensity will be a maximum in the pane of focus.

Width at half peak height is the width, in wavelength units, of thespectral emission curve at half peak intensity. This value may bemeasured as an independent spectral characteristic, or combined withpeak spectral intensity to characterize the spectral emission curve, forexample, the ratio of peak intensity/peak width.

The four curves shown in FIG. 2 illustrate two spectra with relativelynarrow peak widths (curves 10 and 11), and two with relatively broadpeak widths (12 and 13).

Generally peak width increases with increasing size of the PRE, andchanges as the shape of the PRE changes from spherical to non-sphericalshapes in a manner which can be simulated.

Width in the image plane is the halfwidth of the central diffractionregion in the Airy pattern in the image plane. All PRPs aresub-wavelength sources of light, and so their spatial image will be anapproximate point spread function with characteristics defined by theoptical system being used. Assuming that the optical system includes aCCD, with a pixel array of photodetecting elements, the width of thecentral diffraction region, which may cover several pixels, is measuredradially from the peak of the center of the diffraction image to theposition in the center of the image where the intensity has fallen tohalf its peak value (assuming a circular image).

Since the PRPs are subwavelength scatterers, the halfwidth of theintensity pattern as recorded in the image plane will be proportional tothe wavelength of light being scattered. Therefore, for a reasonablysmooth variation in light intensity from a source (such as a Xenon arc),the light is scattered most strongly is at peak intensity, and one canmake a good estimate of peak wavelength by measuring the width of thehalf intensity of the central diffraction region in the image for eachPRP.

As will be seen below, this spectral characteristic is useful forprecise determination of the positions of PREs in a field, andparticularly for determining the distance between two PREs of differentpeak wavelengths that are more closely spaced than the Rayleighresolution distance. The intensity of the peak of the diffractionpattern in the image plane can be used for focusing the detector lens onthe field, with the maximal value giving the best focus.

Polarization measures a spectral characteristic, e.g., peak wavelength,peak height, width at half wavelength, or width at half peak intensityin the image plane, as a function of direction of polarization of lightilluminating a PRE field, or the angle of incidence of polarized light.The polarization characteristic depends on PRE shape rather than size,and is due to the fact that a non-spherical PRE may have more than oneresonance, for example, along the directions of the major and minor axesin an elliptical PRE. In the latter case, illuminating light directedalong the major axis would be shifted toward the red, while thatdirected along the minor axis, would be shifted toward the blue.

Pulse or time response provides a measure of the number of light cyclesof the illuminating light that are required to “pump up” the scatteringto full intensity. PREs have very fast time response (sub-picosecond),and very large pulses of scattered photons can be generated, the onlylimitation being the average input power absorbed. They can acceptpulses between 5 to 500 femtosecond for driving two-photon processes orsecond harmonic generation and other higher order processes.

As noted above, pulsed or timed illumination measurements are generallymade by exposing PREs in the field to short pulses of incident light ofvarying duration, to detect peak wavelength. The time to full resonance,as measured by intensity versus pulse time, also provides a measure ofthe quality of the material as a plasmon resonator. Higher qualitymaterial is characterized by a narrower width of the resonancesignature, a higher peak intensity, and a longer time to reach themaximum intensity of scattering when illuminated by pulses of light atthe peak wavelength.

Phase shift is discussed above in the context of determining spectralpeak at 90 degree phase shift. Phase shift can also give informationabout the response for excitation wavelength away from the resonant peakwavelength.

Fluorescence emission lifetime can be observed in PRE particles havingsurface-localized fluorescent molecules. The fluorescence excitation canbe enhanced by the local electric fields generated near the surface ofthe PRE by light within the plasmon resonance peak. Fluorescenceemission can also be enhanced if the wavelength of the fluorescenceemitted light is within the plasmon resonance peak. Under appropriateconditions, the fluorescence lifetime can be measurably shortened inthis process.

The method can be used to detect changes in the excitation environmentof the fluorescent molecules, e.g., proximate interactions with othermolecules or entities.

Surface enhanced Raman scattering (SERS) relies on the generation ofenhanced electric fields in the region near the surface of a PRE.Interactions between this electric field and nearby materials cansignificantly alter both the scattering characteristics of the resonantparticle and the nearby material. Surface Enhanced Raman Spectroscopy(SERS) traditionally exploits the localized plasmon resonance inroughened or particle evaporated silver films to enhance the Ramanscattering of various materials by as much as six orders of magnitude.The SERS performed in accordance with the present invention is confinedsolely to PREs. In this technique, Raman scattering from the materialsof interest is observed, and the local field generated by the plasmonsis used to enhance the intensity of that scattering by many orders ofmagnitude over traditional SERS. When the Raman active molecule has aresonant absorption near peak of the spectral emission curve of the PRE,the additional SERS enhancement is sufficient to make the Raman signalof the PRE-molecule composite detectable, in accordance with the methodof the invention disclosed in Section III. Measuring changes in the PREresonant Raman spectrum can be used to detect alterations, e.g.,binding, in the local environment of the Raman molecule.

B. Field to be Interrogated

The field that is to be interrogated, in accordance with the method andapparatus of the invention, includes a target or target region having aplurality, i.e., two or more PREs distributed in the target.

The target may be any target that is suitable for viewing by lightmicroscopy, including biological cells or tissues; plant or animal partsor cellular aggregates; a solid surface having surface-localizedligand-binding molecules; a fluid sample containing target analytemolecules, particles or cells; biological sample material, such aschromosomal material placed in an extended condition; artificialmonolayer or bilayer membrane substrates; a microfabricated device, suchas an computer microchip; and a microarray, such as a microarray ofoligonucleotide or oligopeptides on a chip.

Methods for forming PREs and preparing a target having PREs distributedtherein will be discussed in detail below. At this point, three generalcases will be briefly considered. First, preformed PREs are added to atarget, for attachment at specific target sites. The target may bewashed to remove unbound or non-specifically bound PREs. The target maybe manipulated before or after PRE binding to achieve a desiredconfiguration, e.g., an elongated chromosome. Second, nucleation sitesmay be added to the target. After binding to selected locations on thetarget, a metal enhancer solution, e.g., silver solution, is added untilan appropriately sized PRE is formed. In the third case, PREs are formedby photolithographic methods, e.g., photomasking and photoetching, on ametal substrate, e.g., silver substrate.

The types of information which one wishes to determine, by interrogatingthe field containing the target and PREs, in accordance with theinvention include: (i) the total number of PREs of a selected type inthe field, (ii) the spatial pattern of PREs having a selected spectralcharacteristic in the field, (iii) a distance measurement between twoadjacent PREs, particularly PREs separated by a distance less than theRayleigh resolution distance, (iv) a change in the environment of thefield, e.g., dielectric constant, that affects a plasmon resonancecharacteristics, (v) motion of PREs in the field, (vi) whether two PREsare linked, or (vii) a fluorescence or Raman emission of molecules ormaterials attached localized on PREs. Other types of information, arealso contemplated, and will be considered in Sections IV-VI below.

C. Apparatus of the Invention

FIG. 3 is a simplified, schematic view of an apparatus 20 constructed inaccordance with the invention. The target to be interrogated, hereindicated at 22, is supported on a substrate 23 held on a microscopestage 24 which is selectively movable in the x-y plane under the controlof a stage stepper-motor device, indicated generally at 26, under thecontrol of a computer 28, which includes other computational componentsof the apparatus as described below.

The target is illuminated by an optical light source 30 which directsilluminating light, typically light in the visible range, and at one ormore selected wavelength ranges, onto the target surface. As will bedetailed below, the light source typically includes a means 32 forgenerating light of a given wavelength or spectral frequency, one ormore filters, such as filter 34, for producing a desired frequency bandof illuminating light, and a lens system 36 for focusing the light ontothe target, in a manner to be detailed below.

Spectral emission light from the target, in this case light scatteredfrom the target, is directed through lens 56 to an optical detector 38.The optical detector functions, in a manner to be detailed below, todetect one or more spectral emission characteristics of the individualPREs in the illuminated portion of the field. The detector is typicallya CCD (Charge Coupled Device) array which operates to generate and storean array of optical intensity values corresponding to the array pixels,as will be detailed below.

An image processor contained within computer 28 is operatively connectedto the detector to receive values of light intensity at each of thedetector array positions, under each selected illumination condition,e.g., different wavelength or polarization state. The image processorfunctions to construct a computer image of the positions and values ofone or more spectral emission characteristics measured by the detector.Typically, this is done by treating each pixel in the detector array asa position point in the illuminated field, and assigning to each pixel“position” the light intensity value recorded by that pixel. The imagegenerated by the image processor may be a matrix of stored numbers,e.g., position coordinates and associated spectral emissioncharacteristic value(s), or an actual map in which position arerepresented, for example, in an x-y plane, and each measured spectralemission value, represented as a quantity along the z axis, for eachpixel location.

A discriminator 42 in the apparatus, also forming part of computer 28,functions to discriminate PREs with a selected spectral signature, i.e.,a selected range of values of one or more selected spectral emissioncharacteristics, from other light-scattering entities in the computerimage. Examples of the operation of the discriminator will be givenbelow.

C1. Substrate

As indicated above, the target is supported on a substrate which ismounted on a microscope stage. Suitable substrates include standardglass slides, cover slips, clear polystyrene, and clear mica asexamples. Other suitable transparent substrates are those associatedwith a TEM grid, including for example, formvar, carbon and siliconnitride. These TEM-associated substrates are all optically transparentat the thicknesses used. Conducting, semiconducting, and reflectingsubstrates are also suitable for PRE applications.

Another suitable substrate for use in the present invention are thosewhich may initially appear opaque to the spectral wavelengths ofinterest for PRE observation, but which can be rendered suitable by theapplication of a suitable fluid or vapor. An example is whitenitrocellulose “paper” as used for the transference of biologicalsamples of interest in diagnostic techniques such as “Southerns”,“Northerns”, “Westerns”, and other blotting, spotting, or “dip stick”tests. Once the materials of interest have been transferred and fixed asdesired, the PRE's can be applied as preformed entities, or one canapply PRE nucleation entities and enhance as described below. The whitenitrocellulose at this stage may typically present significantnon-specular light scattering which makes it difficult to visualize thePREs. However, if a suitable treatment which results in a significantreduction of the non-specular scattering is used, for example, allowingacetone vapor to encompass the nitrocellulose substrate, whilemonitoring the PREs, the substrate can become much less opaque, andpermit efficient observation of the PREs.

Silicon is a preferred substrate for many PRE detection applicationsbecause it can be made very smooth and free of defects, resulting invery little non-specular scattering under darkfield illumination. Oneexample of a particularly preferred silicon substrate is the highlypolished, etched, and defect free surfaces of silicon wafers commonlyused in the manufacture of semiconductors. The nearly complete absenceof contaminants and surface imperfections of such a substrate producesexcellent contrast of the PRE scattering under darkfield illuminationconditions. However, it should be appreciated that such silicon waferstypically have a thin layer of SiO₂ present on their surface as a resultof the various processing steps. It may be mentioned that siliconsubstrates with approximately 100nm or more of SiO₂ on their surfaceproduce some of the most intense, high contrast PRE spectra so farobserved from a solid substrate, and it may be advantageous tointentionally grow a sub-micron layer of SiO₂ on the silicon wafersurface.

If the oxide layer is removed from the silicon surface in a manner thatprevents rapid re-growth of an oxide layer, for example, by etching inHF acid and passivating the surface with hydrogen, the optical image ofthe “point-source” PREs has been observed to be torus-shaped, ratherthan the usual Airy ring pattern with a bright central region. This“doughnut” phenomenon most likely arises as a result of damping of thetransverse driving electric fields (those parallel to the siliconsurface), leaving only the perpendicular driving fields which can excitea plasmon mode that radiates well, but not at all directly along thenormal. This property of bare silicon substrates can be useful indetermining whether a particular PRE is closely bound to the surface ofthe silicon substrate, or is bound via a tether molecule or system thathas placed it further from the surface, thereby changing the dipolecomponent scattering ratios.

C2. Light Source and Detector

With continued reference to FIG. 3, light-generating means 32 in thelight source may suitably be a mercury, xenon, or equivalent arc; or aQuartz-tungsten halogen bulb, of approximately 20 to 250 watts, whichprovides incident light in a frequency band corresponding to wavelengthsfrom approximately 350 nm to 800 nm, for visible light PRE scattering,or a conventional UV source for lower-wavelength PRE scattering.

Filter 34 typically includes a set of pre-selected narrow bandwidthfilters, allowing manual or computer controlled insertion of therespective filters. The bandwidth for such filters is typically 5-10 nm.

Other methods of illuminating a target with a series of selectedbandwidths include the use of light sources such as lasers of all typeswhere one may utilize very narrow bandwidths. Multiple frequency sourcesare also contemplated, such as tuned lasers (i.e. Ar-ion) to select anyof the characteristic defined strong “line” sources. Alternatively agrating or prism monochrometer can be used. All the light sources can beeither of continuous or pulsed variety, or a suitable light amplitudemodulation device (not shown) can be inserted in the incident path tovary the intensity level in a prescribed temporal manner. Thepolarization of the light to be incident upon the sample can be variedby the insertion of suitable filters or other devices well known to theart.

The microscope in FIG. 3 is illustrated to be configured with anepi-illumination system, whereby the collimated light from the sourcefollowing filtering as desired impinges onto a half silvered mirror 38,and is reflected downwards towards the Darkfield/Brightfield (DF/BF)lens 40. In this particular type of DF/BF application, the incidentlight that would have had rays passing through the objective lens isphysically blocked by an opaque circle 42, which is suspended by veryfine webs 44, so as to allow only a concentric band of light to passsuch as bounded radially and illustrated by the rays 46. The unitcomprising mirror 38 and opaque circle 42 may be built into anadjustable block 48 that can be manually (or robotically) moved therebyconverting the microscope from DF/BF to alternate forms of operation.

Light reflected from the mirror may in turn be refracted or reflected(by a suitable circular lens element 50, fixed to the objective lensmount into a hollow cone of incident light 52, converging toward a focusat the sample plane of the target. As previously noted, the specularreflection of such rays causes them to return along the lines of theincident cone trajectories, where they are ultimately absorbed orotherwise removed from the optical system.

In this darkfield system illustrated in FIG. 3, the angle between theoptic axis and the incident rays illuminating the sample is larger thanthe largest angle between the optic axis and the rays scattered by thePREs which is accepted into the objective lens element 45, which isillustrated to be of the refractive form. Also incorporated in the totaloptical microscope, although not shown, is the ability to divert thelight rays away from detector 38 to other ports whereby the image may beobserved visually through standard binocular eyepieces, or to yetanother port, for example, for photographing the illuminated field.

It has been found to be suitable to use a Nikon DF/BF lens model CF PlanBD ELWD with magnification 100× and numerical aperture (N.A.) 0.8 as thelens system 54, and also a model CF Plan BD ELWD with magnification 20×and N.A. 0.4. In that case, the rays entering the objective element ofthe lens may be rendered parallel and incident upon the 50% mirror 38,and into a relay lens 56 (typically magnification of 2×or 5×) that focusthe rays to an image plane on detector (image capture device) 58, wherethe detection is performed by a suitable CCD camera system.

The optical system, including lens 56, is preferably constructed toproject the field being viewed into an area corresponding to the arrayof the detector, so that each pixel in the array is reading light from adefined region of the field.

Various image capture devices known in the art may be used, includingfiber coupled photo-diode arrays, photographic film, etc. One exemplarydevice is a thermoelectrically cooled CCD array camera system, modelCH250, manufactured by Photometrics, of Tucson Ariz. This deviceutilizes a CCD chip model KAF1400, having a 1032 by 1037 pixel array.

It will be appreciated that the detector serves to detect a spectralemission characteristic of individual PREs and other light-scatteringentities in the field, when the field is illuminated by the lightsource, simultaneously at each of the regions in the field correspondingto array pixels.

C3. Image Processing Discrimination and Output

Where the detector is used, for example, to detect spectral peakwavelength, peak intensity, and/or half width of the spectral peak, thedetector measures light intensity at each of a plurality of differentilluminating light frequencies, simultaneously for each of the fieldregions corresponding to a detector array pixel.

The emission (scattering) values measured at each frequency are stored,allowing spectral emission curves for each region to be constructedafter a full spectrum of illumination. From these curves, peakwavelength, peak intensity, and width at half intensity are calculatedfor each region. Similarly, the peak halfwidth in the image plane can bemeasured with a CCD array as described above.

The detector may be supplied with comprehensive software and hardwarethat allows timed exposures, reading out of the pixels into suitablefiles for data storage, statistical analysis, and image processing (asone of the functions of computer 28). This capability serves as an imageprocessor for constructing from signals received from the detector,first the values of the spectral emission characteristic(s) beingdetermined, and then a computer image of these values and thecorresponding associated field positions.

The image constructed by the image processor may be a matrix of storedpoints, e.g., a matrix of associated values of each field position(regions in the field) and values for one or more measured spectralcharacteristics, or may be an actual map of field positions, e.g., inthe x-y plane, and associated spectral emission values in the z plane.

The computer in the apparatus also provides discriminator means fordiscriminating PREs with a selected spectral signature from otherlight-scattering entities in the computer image. The basis for thisdiscrimination is noted above in the discussion of various spectralemission characteristics and their correlation with physical propertiesof light-scattering entities.

Thus, for example, to discriminate PREs with a selected spectral peakwavelength and peak width at half intensity, the computer imagegenerated could provide a matrix of all field regions and the associatedspectral peak wavelength and width values. The discriminator would thenselected those regions containing PREs whose spectral signature meetscertain ranges of these two spectral emission values. Depending on theparticular values chosen, the discriminator could classifylight-scattering entities in the field in a number of ways, includingdistinguishing:

1. PREs with a selected spectral signature from all otherlight-scattering entities in the field;

2. PREs from non-PRE light scattering entities in the field;

3. For a selected type of PREs, those selected PREs which areinteracting with one another and those which are not; and

4. One selected type of PRE from another selected type of PRE in thefield.

In each case, the basis for the discrimination may be based on detectedvalues, for each light-scattering entity in the field, of peak position,peak intensity, or peak width at half intensity of the spectral emissioncurve, peak halfwidth in the image plane, and polarization or angle ofincidence response. Other spectral characteristics mentioned above arealso contemplated. In particular, where the PREs have surface-localizedfluorescent molecules or Raman-active molecular entities, the detectingmay detecting plasmon-resonance induced fluorescent emission or Ramanspectroscopy emission from one or more of said molecules or entities,respectively, and these values are used as a basis of discriminatingsuch PREs from other light-scattering entities. FIG. 11 shows a typicalRaman spectrum of a Raman-active molecule carried on the surface of aPRE.

The information obtained from the discriminating step is then used toprovide information about the field. Various types of informationavailable are discussed in Sections IV-VI below. Among these are:

1. The total number of PREs of a selected type in a field. Here thediscriminating step includes counting the number of PREs having aselected range of values of a selected spectral emission characteristicin the constructed computer image;

2. Determining a spatial pattern of PREs having a selected range ofvalues of a selected spectral characteristic in the field. Here thediscriminating includes constructing an image of the relative locationsof PREs with those spectral-characteristic values;

3. The distance between two adjacent PREs, particularly where thisdistance is less than the Rayleigh resolution distance. Here thedetecting includes exposing the field with light of one wavelength, toobtain a diffraction image of PREs in the field, exposing the field withlight of a second wavelength to obtain a second diffraction image ofPREs in the field, and comparing the distance between peaks in the twodiffraction patterns;

4. Interrogating a change in the environment of the field. Here thediscriminating includes comparing the values of the detected spectralcharacteristic of a PRE in the field before and after the change, e.g.,change in the dielectric of the field;

5. Detecting motion of PREs in the field. The detecting here includesdetecting the centers of the diffraction patterns of the PREs in theimage plane, as a function of time.

C4. Other Embodiments

Simultaneous imaging of even 100 PRPs or more in the illuminated fieldmay be readily and efficiently accomplished, using the apparatus justdescribed. Alternatively, the apparatus may be designed to “read” aspectral characteristic of each PRE in a field by sequentially scanningeach region in a field with a focused-beam light source, and/orsequentially detecting light scattering from each region in the field,by moving the microscope stage through a small interrogation regiondefined by stationary optics, sequentially interrogating each region todetermine values of a selected spectral characteristics, according toabove-described methods.

For detecting fluorescent images, the light source is filteredappropriately for the excitation spectrum of the desired fluorophore,and a suitable filter (not shown) is placed in the region between mirror38 and relay lens 56. This filter is chosen to substantially block theexcitation light and permit passage of light in a band matching theemission spectrum of the fluorophore(s) of interest.

The value of the ability to make comparison of the multiple images ofdarkfield PRP treated, brightfield dyed, and/or fluorescent stainedsamples such as cells and other entities of interest to biological andmedical researchers and clinical applications can be readilyappreciated.

There are several suitable means for bringing in the incident light soas to establish effective darkfield conditions in conjunction withsuitable means for preferentially and efficiently observing the lightscattered by the PREs. For transparent substrates the incident light maybe brought in either in transmittance through the substrate, reflectancefrom the “objective side surface”, or via TIR (total internalreflection) at the interface near which the PREs are situated, as shownin FIG. 3 and described in more detail below. In the latter case theevanescent tail of the TIR light may also be used to excite the PREs, ifthey are directly outside the reflecting interface, even though suchlight field distributions do not radiate directly to the objective lens.

For non-transparent substrates, the light must be incident in a mannerthat results in as near specular reflection as possible, with a minimumof such light reaching the objective. There are several means foraccomplishing this condition. As one example, incident light may berouted to the field of view through one or more optical fibers. Thefibers may be oriented such that light reflects off the substrate at aglancing angle and does not enter the objective lens of the microscopesystem used to image the PREs. It is also advantageous to usecommercially available DF/BF objective lenses. Examples are those soldby the Nikon Company, Long Island N.Y. An example of the way such aDF/BF lens may be used is illustrated in FIG. 3.

The objective lens of system in the apparatus can be made as either areflecting or refracting lens. For certain PRE applications, especiallythose requiring the most accurate and rapid focusing of the objectivelens as a function of light wavelength, reflecting lenses may bepreferred because chromatic aberrations can be greatly reduced.Refracting lenses, even those carefully made to compensate for chromaticaberrations when observing light emitted by PREs, can still exhibitsignificant deviation in their frequency dependence of the focal length.The fact that the PREs near the peak of their plasmon resonance can beof such extraordinary brightness for a sub-wavelength sized sourceallows them to be utilized for evaluation of optical components, wherebyone can observe a variety of deviations from the component's ideal“point source” response.

The use of particular lens types which enhance the numerical aperture ofthe objective is also contemplated. PREs can be imaged with a standard“solid-immersion” lens having a spherical top and flat lower surface.Another such contemplated lens is a liquid analog to a solid immersionlens (SIL) having a fluid between the lower surface of a truncated solidimmersion lens and a substrate which has an approximately equal index ofrefraction as the lens material.

Such a lens is illustrated in FIG. 4. The lower flat surface 80 of thelens is cut shorter than a standard solid immersion lens. With the indexmatched fluid 82 between the lower surface 80 and a substrate 84, thefocal plane of the lens is at the usual r/n (where n is the index ofrefraction of the lens material) location, which is now inside the indexmatched fluid. PRPs in the fluid at this focal plane are thus imagedwith this system, allowing focusing on non-substrate bound PREs insolution. Such a lens can readily be added to a commercial DF/BFmicroscope lens by one of ordinary skill in the art, thereby resultingin an improved numerical aperture and increased magnification forviewing samples in a liquid matrix. In particular, this modificationresults in increased brightness and clarity in visualization of thePREs. This configuration allows the user to vary the focal position in aliquid sample while retaining a large numerical aperture and removes theusual requirement of total internal reflection-based illumination as isused for the SIL. This system therefore allows the monitoring ofmovement of a particular PRE in solution.

In other preferred embodiments, darkfield optics are chosen to optimizethe signal to noise ratio of the PRE signal. This often includesimproving the contrast by reducing the background (non-PRE) scatteredlight to a minimum, or to a minimum relative to the amount of PREscattered light observed. For example, using ordinary 1″×3″ glassmicroscope slides, it was determined that the Nikon DF/BF lenses of theextended working distance class had a better contrast for observationusing the 0.8 NA lens compared to the 0.9 NA lens at the samemagnification level (100×), despite the fact that the higher the NA of alens the more scattered light is collected from the PRE. This may beattributed to the change in reflecting properties of the bare glasssubstrate since the angle of incidence of the darkfield illuminationlight is changed when the NA of the objective is changed.

Polarized incident light can also be used at specific angles (i.e. theBrewster angle) to reduce the amount of reflected light from the surfaceof the substrate. This improves contrast by reducing the amount ofnon-PRE scattered light which enters the objective lens. When imagingnon-spherical PREs such as ellipsoidal PREs as described above, theresponse to plane polarized incident light can also be used todistinguish different PRE populations.

In another embodiment, a transparent substrate is used which issufficiently thick so as to reduce the amount of light scattered fromits bottom surface that reaches the detector. If particulates arepresent on the bottom surface of a thin glass substrate, some of thescattered incident light will re-enter the detection system and increasebackground. By increasing the thickness of the substrate, one displacesthe region of the spurious scattering further from the optical axis,thereby reducing the amount of non-PRE scattered light that enters thedetector.

Total internal reflection (TIR) may also be used to illuminate PREs in atransparent substrate from beneath. The evanescent tail of such lightcan be effectively used to excite the PRE located near that interface.There are several methods for exciting PREs with totally internallyreflected light, such as using an optical fiber whose dimensions andindices of refraction of the inner core and outer layer are chosen sothat there is sufficient evanescent field at the fiber outside surfaceto excite PREs placed thereon. The light emitted from the PRE can betransferred back to the fiber, forming a reflected source of light whichcan be observed by standard methods.

In another embodiment, illustrated in FIG. 5, a Dove prism 70 isilluminated from the side. A standard glass slide 72 is placed on thetop surface of the Dove prism 70, with a suitable index matching oil 74in between. The incident light 76 is brought in parallel to the majorsurface of the prism 70 and is refracted up toward the slide 72. Theangle of incidence at the slide is selected to be greater than thecritical angle, and results in total internal reflection at the uppersurface of the slide 72. The light then exits the other side of the Doveprism, again parallel to the major face of the prism 70. Evanescentelectromagnetic fields excite PREs bound to the upper surface of theslide, and emit the usual plasmon resonant scattered light into anobjective lens located above the slide.

This Dove prism geometry is convenient for bringing light from diversesources such as laser, quartz halogen, arc lamp and the like via anoptical fiber or lens to impinge upon the side of the Dove prism, andfilters may be conveniently interposed therebetween to further controlthe nature of the incident light.

The DARKLIGHT(™) light source from Micro-Video of Avon, Mass. can alsobe used in a total internal reflection illumination system, although ithas been found generally inferior to the Dove prism embodiment describedwith reference to FIG. 5. This totally internally reflecting slideilluminator takes light from a halogen source into an optical fiber,then into the edge of a glass slide. The light undergoes total internalreflection numerous times while spreading down the slide, exciting PREswith evanescent fields as with the embodiment of FIG. 5. This system isdescribed in detail in U.S. Pat. No. 5,249,077. Additionally, PREs canbe observed via the illumination described in U.S. Pat. No. 3,856,398.

Oil inmmersion lens systems can also be used in conjunction with TIRillumination. In these systems, though, the index of refraction of themedium for internal reflection must be greater than that of the oilbeing used. Accordingly, flintglass, having an index of refraction of1.7, is one preferred prism material.

FIG. 6 illustrates in cross section a DF/BF objective lens systemcomprising a reflective objective lens 90 combined with a suitableenclosing darkfield illumination ring element 92 as an alternativeillumination scheme. Although the use of such a two component reflectivelens reduces the intensity at the center of the diffraction pattern, thefact that it also substantially reduces the chromatic aberrationcompared to the refractive lenses described in the prior art, makes it apreferred embodiment in conjunction with the means for obtaining thesimultaneous frequency dependent scattering data for a multiplicity ofPREs and for the other non-PRE scattering entities within the field ofobservation that may be imaged and which need to be distinguished andrejected for many applications.

In some applications, optical microscopy methods must be tailored toboth optically image and analyze the PREs and also to observe the samePREs and associated sample material with additional instruments such asone or more forms of electron microscopy. In these cases, darkfieldoptical microscopy must be performed with substrates suitable forelectron microscopy as well. Because the electrons must pass through thesample and substrate, the substrate must be very thin, typically wellunder 1 μm. Common substrates include formvar and/or carbon depositedupon a supporting grid. Background scattering is reduced from the gridboundaries or “bars” by arranging for the field of application of theincident darkfield illumination to be within the spacing of the grid“bars” and/or to restrict the field of view of the collecting objectivelight for the part of the sample under observation. For the BF/DFobjective and microscope system, grids with a spacing of up to 400 barsper inch and silicon nitride membranes are especially suitable.Alternative forms of darkfield illumination include a separate opticalfiber or lens with darkfield illumination from below the grid substrateand observation of the scattered PRE light from above with a BFobjective lens.

IV. PRP Compositions

The invention further includes a suspension of plasmon resonantparticles (PRPs) having one or more populations of PRPs. The compositionhas four distinguishing features: (i) the PRPs in each population have aspectral width at halfheight of less than 100 nm; (ii) the PRPs in asingle population are all within 40 nm, preferably 20 nm of a definedspectral emission peak wavelength; (iii) at least 80% of the PRPs in thecomposition are in one or more of the populations and have a spectralemission wavelength in one of the three ranges >700 nm, 400-700 nm; and<400 nm; and (iv) each population has at most a 30% overlap in number ofPRPs with any other population in the composition.

The first feature addresses the quality of the PRPs, high-qualityemitters being characterized by a relatively narrow frequency range ofscattered light. The second feature provides homogeneity of spectralemission properties for all PRPs within a given population.Specifically, PRPs within a given population all have a peak wavelengthwithin 40 nm of a defined wavelength. The third requirement providesthat a large majority of the PRPs (at least 80% by number) are in one ofthree different wavelength ranges. The fourth requirement defines theuniqueness of the populations, assuming that each population has adistribution of spectral peak wavelengths within 40 nm of a given peakwavelength. The two distribution curves can be no closer than thedistance at which 30 number percent of the particles in one populationfall within the distribution curve defined by an adjacent population.

Typically, the PRPs in the composition are in one or more of thepopulations, all in the 400-700 nm wavelength range. The PRPs may behomogeneous, e.g., all blue particles, or may be in one of morepopulations, e.g., discrete populations of red, green, and blueparticles, collectively making up 80% of the PRPs in the composition.Particles in this spectral range may be formed, as described below, assolid silver particles, silver particle with a gold core, or particleswith a dielectric core and an outer silver shell of at least about 5 nm.

In one general embodiment, particularly for use in a variety ofdiagnostic applications, the particles have localized at their surfaces,(i) surface-attached ligands adapted to bind to ligand-binding sites ona target, (ii) fluorescent molecules, and (iii) Raman-active molecularentities. The ligands are one of the members of a conjugate pair thatcan include antigen/antibody, hormone/receptor, drug/receptor,effector/receptor, enzyme/substrate, lipid/lipid binding agent andcomplementary nucleic acids strands, as examples.

The PRPs in the composition may have different surface-localizedmolecules on different groups of PRPs. These different groups may bedifferent PRP populations, that is, PRPs with different spectral peakwavelengths, or may be localized on PRPs in a homogeneous PRPpopulation.

For use in identifying a target having first and second ligand-bindingsites, the different surface localized molecules may be differentligands effective to bind to different ligand-binding sites, such as twodifferent-sequence oligonucleotides that bind at different sequenceregions of a common target polynucleotide, or two different ligands thatbind to different ligand-binding sites on a macromolecular target.

PRPs may be formed made by a variety of known methods, includingcolloidal chemistry, soluble gel, evaporation/annealing,nucleation/growth via an enhancer, autoradiography, and photoreaction insilver halides and other materials via electromagnetic energy in theform of light, X-rays, or other incident wavelengths. In addition,lithography, electrodeposition, electroplating, and sputtering with ascanning tunneling microscope (STM) tip can be used.

Where the PRPs have surface localized, e.g., attached ligands, the PRPsare able to bind selectively to a target of interest which carries theother half of the ligand/ligand-binding conjugate pair.

As indicated above, it is also advantageous to produce population ofPRPs having different defined peak wavelength values. When definedpopulations of PRPs are combined with specific binding characteristics,a new class of sub-microscopic probe is created which has significantadvantages over all currently used labeling techniques. The PRPformation techniques described below may be used to create such probes.

A. Formation of the PRPs By Metal Enhancement

In this method for forming PRPs, a nucleation center, typically a metalnucleation center in the 1-20 nm size range, is placed at a targetedlocation, followed by in situ development (enhancing) of the fullconductive body of the PRP. In some applications, a spatiallypre-specified position is designated for the placement of the nucleationcenter. This is in contrast with other applications where nucleationcenters are used to probe a matrix for a particular substance or featurewhose position is not known. Pre-specified placement may be advantageousin metrology and/or instrumentation applications which are furtherdescribed below. For example, it may be desired to place a single PRP onthe end of an optical fiber or a scanning microscope tip. Also, it maybe desired to create a pattern of PRPs in a particular geometricconfiguration.

Because it is often desirable to create PRPs with controlled spectralcharacteristics, the enhancing may advantageously be performed whilebeing monitored, stopping the process when a PRP having some pre-definedspectral characteristic is formed. This is especially convenient whenenhancement chemistry which is not affected by light is used. It is alsopossible to monitor PR formation by periodically terminating theenhancement process, observing the characteristics of the entity orentities formed, and re-initiating the enhancement process if one ormore spectral characteristics such as color, for example, are not withina desired range.

The nucleation center is typically a gold particle 1-10 nm in diameter,and the metal used for enhancing this nucleating site to PRP size issilver. However, other elements including, for example, platinum,nickel, and palladium, and macromolecules, such as polynucleotidemolecules, are also contemplated as nucleation centers for thesubsequent enhancement process. Non-metallic materials may also be usedas nucleating centers, such as protein and nucleic acid.

The configuration of the nucleating center can be controlled so as toproduce a PRP having a desired shape or characteristic. For instance,triangular or ellipsoidal regions of nucleating material can be formed.The deposition process may involve many metal deposition techniquesknown in the art, such as vapor deposition, sputtering, Ga-focused ionbeam (FIB) milling of the thin film prepared of the desired material,and electroplating into nanopores. Particularly well controlledplacement of nucleating material is possible by discharging nucleatingmaterial from the metal tip of a scanning tunneling microscope.Techniques have also been developed whereby individual metal atoms arepicked up with the tip of a scanning tunneling microscope, moved, andput down at a desired location. Individual atoms may also be “pushed” toa desired location with a scanning tunneling microscope tip. Thistechnique may be used to place, for example, 10 gold atoms at aspatially pre-specified position for use as a nucleation center.

In one embodiment, PRPs or PRP nucleating centers are placed at adesired location by introducing the PRP or PRP nucleating center into adrawn micropipette tip, moving the PRP or PRP nucleating center to adesired location and depositing the PRP at this location using standardmicro-manipulation techniques. The pipette tips are filled with thedesired material in solutions of varying viscosity (i.e. with gelatin orother matrices), then the tip is manipulated to a selected locationwhile observing under the microscope. By applying pressure to thesolution in the pipette, very small quantities can be deposited onto adesired substrate. Other micromanipulation techniques are also possible.

Because the PRP material will have a positive dielectric constant atfrequencies above the resonant frequency, the metal particles can beoptically trapped in a focused light beam of appropriate wavelengthusing principles similar to currently practiced optical trapping ofplastic particles. The PRP can thus be placed at a spatiallypre-specified position by manipulating the light beam in which it istrapped. Channels of gel may also be created in order to route PRPs tospatially pre-specified positions electrophoretically. If the channel isconfigured to pass proximate to a designated location, a PRP in thechannel will move under the influence of an electric field so as to beguided to the location. Electrostatic bonding techniques can also beused to removably attach PRPs to spatially pre-specified positions.

In one embodiment, an array of one or more conductive pads ofapproximately 10 to 100 microns in diameter may be created usingstandard integrated circuit lithographic techniques. Each pad mayfurther be selectively connected to a voltage source. As PRPs insolution can be negatively charged, applying a suitable potential to apad will attract a PRP from solution and onto the conductive pad.Removing the voltage and reversing the sign of the applied potential mayfree the PRP if the surface binding forces between the PRP and the padare weak, i.e., approximately one picoNewton.

Conjugated nucleating centers and/or conjugated PRPs can also be placedat a spatially pre-specified position by immobilizing the other half ofthe conjugate pair at the spatially pre-specified position and bindingthe conjugate on the nucleating center or PRP to the other half of theconjugate pair.

Individual nucleating centers (or fully formed PRPs) may also bedeposited by ejecting droplets of metal particle containing fluid to asubstrate and having the fluid evaporate away by techniques analogous tothose used in ink-jet printing. In this technique, one or more metalparticles can be delivered to a specific location defined by the dropposition. In some embodiments, the concentration of PRPs or nucleatingcenters in the fluid is chosen so that it is statistically likely forone or no PRP or nucleating center to be contained in each drop. Ifnucleation centers are deposited, they may be subsequently silverenhanced to form PRPs. It may further be noted that these techniques canbe used to create a desired geometric pattern of PRPs. When making sucha pattern, drops can be redeposited at those locations where no PRP wasejected during the first pass. Articles with such patterns of PRPs canbe useful in object identification, semiconductor mask registration, andother uses as are described in more detail below.

As defined herein, the term “in situ” indicates that the PRP is bound toa substrate, immersed in a solution or suspended in a matrix. Thedefinition of “substrate” is discussed below and includes any entitywith which a PRP can associate such as tissue sections, cells, TEM gridsmade from, for example, formvar, silicon (including silicon nitride andsilicon dioxide), mica, polystyrene, and the like. These nucleationcenters are typically colloidal gold preparations, and suitablepopulations of nucleation centers are commercially available either infree form or attached to various biological molecules.

The silver enhancement of gold nucleating centers to produce largersilver masses which can be visible under an electron and/or a lightmicroscope is currently practiced, and some procedures for imagingbiological systems using silver enhanced gold particles have beenextensively developed. Reagents for performing the enhancement, as wellas the nucleating centers themselves, are accordingly commerciallyavailable. Antibody bound gold nucleation centers are available fromseveral sources, including E-Y Laboratories of San Mateo, Calif., andAmersham of Arlington Heights, Ill. Furthermore, a large body ofliterature describes a variety of suitable methods for performing suchenhancement (see, for example, M. A. Hayat, Ed., Immunogold-SilverStaining—Principles, Methods, and Applications, CRC Press, 1995, thedisclosure of which is hereby incorporated by reference in its entirety,most particularly Chapters 2 and 7, which describe several experimentaltechniques for silver enhancing gold nucleation centers and a discussionof the silver masses formed thereby).

The enhancement process is preferably performed by mixing goldnucleation centers with a solution of a silver salt such as silveracetate, silver lactate, or silver nitrate, and a reducing agent such ashydroquinone in a citrate buffer at a pH of approximately 3.5 to 3.8. Ithas been suitable to provide a concentration of approximately 6 mM forthe silver salt and approximately 33 mM for the hydroquinone.Commercially available silver enhancement solutions containingappropriate quantities of silver and reducing agents are alsocommercially available from, for example, BBI of the United Kingdom.Those of skill in the art will appreciate that choice of bufferingsystem, silver salt, reducing agent, and other enhancement parametersare preferably optimized for the local environment, target locations,and the like, and that such optimization can be performed without undueexperimentation.

In accordance with one aspect of the present invention, PRPs useful inthe invention are prepared by silver enhancing nucleating centers untilthe particles possess the properties of plasmon resonant particles. Mostpreferably, the silver enhancement parameters are controlled such thatthe PRPs created have pre-defined spectral characteristics, such asappearing a particular desired color when viewed with darkfieldmicroscopy. During enhancement, spectral characteristics may be observedfor one individual evolving PRP or simultaneously for a plurality ofindividual PRPs. Thus, PRPs having specific physical properties can bemade and placed at a desired location. In addition, PRP nucleatingcenters can be placed at a desired location, either in situ, in vitro orin vivo, followed by silver enhancement to produce a PRP at the specificlocation.

B. Specific Examples of PRP (or PRE) Formation by Metal Enhancement

PRPs were prepared by silver enhancement of a gold nucleating center asdescribed in the following examples.

EXAMPLE 1 Placement of Gold Colloids on a Substrate

The Alcian Blue method is one method for attaching gold colloidnucleating centers to a substrate by chemically treating one or morespatially pre-specified positions of the substrate. Alcian blue is apositively charged dye which promotes adhesion of the negatively chargedgold colloids by charge interactions. Portions of the substrate at whichPRPs are undesired may be coated with a blocking agent such as BSA. A100 microliter drop of 100:1 dilution of 5% acetic acid and 2% alcianblue was placed onto a carefully cleaned glass slide for ten minutes.The active site of the substrate was then immersed in doubly distilledwater, rinsed, and dried.

The gold colloids were diluted to the desired concentration. Thesolution was placed on the alcian blue treated region of the substrateand incubated for 10 minutes. The substrate was then rinsed withdistilled water.

The gold colloid attached to the substrate was enhanced as described inExample 2.

EXAMPLE 2 Silver Enhancement of Individual Sold Nucleating Centers

One ml of a 0.1 mg/ml solution of gelatin was mixed with 50 μl initiatorand 50 μl enhancer in an eppendorf tube. The initiator and enhancer wereobtained from BBI International (United Kingdom) silver enhancement kit,light microscopy (LM) version, catalog No. SEKL15. The substrate wasimmediately covered with the enhancer solution and timing was started.The substrate was then viewed under darkfield illumination to determinewhether PRs were present. The approximate enhancement time for colloidson glass, silicon or TEM substrates was about one minute, while theapproximate enhancement time for conjugate colloids attached tobiological substrates was about seven minutes. These times weredetermined by continuously observing the scattered light from anindividual evolving PRP under darkfield microscopy while the enhancementwas taking place. It can be appreciated that precise enhancementdurations can be utilized to control the scattering response, andtherefore the color, of the PRPs created. Enhancement was stopped byrinsing thoroughly with distilled water.

EXAMPLE 3 Generation of PRPs in Solution

For uncoated colloids, 100 μl stock gold colloid (BBI International,United Kingdom) was added to 20 ml gelatin solution (0.1 mg/mil). Forprotein conjugated colloid, 100 μl stock conjugated gold olloid wasadded to 20 ml doubly distilled water with 1% bovine-serum-albumin (BSA)to block the surface. Conjugated colloids used were bovine serumalbumin, goat anti-biotin and rabbit anti-goat IgG. Typical gold colloidconcentrations are:

For uncoated colloid:

3 nm stock—˜3×10¹³ particles/ml

10 nm stock—˜5.7×10¹² particles/ml

20 nm stock—˜7×10¹¹ particles/ml

For protein conjugated colloid:

1 nm stock—˜2×10¹⁵ particles/ml

5 nm stock—˜1.7×10¹⁴ particles/mil

10 nm stock—˜1.7×10¹³ particles/ml

20 nm stock—˜2×10¹² particles/mil

Three drops (150 μl) initiator was then added. Ten μl increments ofenhancer were then added while stirring, until the amount correspondingto the desired PRP scattering peak wavelength was added. The PRPresonance was checked by darkfield measurement or by absorptionspectroscopy as described previously.

C. PRP and PRE Formation With Lithography and Illumination

Lithographic techniques can be used to specify where a PRP (or PRE) willbe formed and to control its shape, shape, morphology and composition.Both positive and negative resist methods can be utilized to lay downeither nucleation centers or fully formed PRPs. In the positive resistmethod, portions of a layer of resist is removed in order to form moldsinto which metal is deposited. After such deposition, resist and extrametal is lifted off, leaving a nucleating center or a fully formed PRPbehind. In the negative resist method, a chosen layer of silver or othersuitable metal is covered with a layer of resist. Portions of thisresist layer are then polymerized. When nucleation centers are formedwith this technique, the characteristic size of this polymerized regionmay advantageously be approximately 5-20 nm. For laying down PRPs whosepeak wavelengths are in the optical spectrum, the characteristic sizesof the polymerized regions are advantageously 40-125 nm. Silver andun-polymerized resist are then etched away, leaving the metal nucleationcenters or fully formed PRPs under the polymerized portions of theresist. As another alternative, metal forms can be produced which arelarger than desired, and material may be etched away by ion millinguntil a PRP of desired characteristics is formed. All of thesetechniques are used in the electronics and other industries and are wellunderstood by those of skill in the art.

In addition, metal salts and halides (i.e. in film) can be irradiated toobtain nucleation centers or entire particles. Enhancement can beperformed with the techniques set forth above, or can also be performedby thermally annealing the metal particles, or may be performed as isdone in photographic development processes, wherein a film ofphotochemical metal salts or metal halides is locally irradiated withlight until PRPs are produced, and the film is then fixed and developed.There are several ways a localized light spot may be produced forforming nucleating centers or PRPs at desired locations. In oneembodiment, a very localized light spot can be generated using a metalside-coated tapered fiber “near-field” scanning tip to help confine thenucleation to a suitable small size or to grow the silver grainsdirectly. Alternatively, a pre-made special optical fiber tip ofpreferably approximately 150 nm diameter having a PRP on its endconcentrates the light at a desired location to “write” these nucleationcenters onto a photosensitive surface material. In another embodiment, asolid-immersion lens may be utilized to focus the light onto the metalsalts or halides. The solid-immersion lens may include a PRP at itsfocal point in order to further intensify the local radiation of thesubstrate.

Photochemical silver salts or halides are also sensitive to electron andion beam irradiation, as well as irradiation from radioactive elements.It will be appreciated that these photographic methods can also be usedto produce arrays or patterns of PRPs of desired configuration.

Whether the developing PRPs are in solution, or bound to a substrate,the enhancing process can be observed in situ with darkfield microscopyand the process stopped once the PRP has reached the desired size whichcorresponds to a particular color. During light sensitive enhancementprocedures, the progress of the enhancing process can be observed bywashing out the enhancer, observing the light scattering properties ofthe particles created, and re-initiating enhancement until PRPs withdesired spectral characteristics are obtained. In an alternativeembodiment, a relatively light insensitive enhancer can be used and theenhancing process can be observed under continuous darkfieldillumination and scattering data collection. Of course, once specificprotocols have been developed which indicate enhancer amounts,incubation times, etc., to produce PRPs with given properties,observation of the enhancement process becomes unnecessary.

D. Formation of a Conjugated PRPs

As is shown in Example 3 above, it is possible to enhance nucleatingcenters which are bound to a biological macromolecule such as anantibody. This enhancement of conjugated gold antibodies can besuccessfully performed even when the conjugated gold/antibody is inaqueous solution and has not been previously bound to an antigen in acell or cell organelle. Surprisingly, it has been found that aftersilver enhancement of free conjugated gold nucleating centers to createPRPs, an appreciable fraction of the conjugate molecules originallypresent on the gold colloid are surface bound to the resulting PRPs.Furthermore, the biological molecule can retain biological activityafter the enhancement process.

In conjunction with this method, the amount of bound conjugate on agiven PRP can be controlled by controlling the size of the conjugatebound nucleation center. For example, a commercially available 1 nmdiameter nucleation center may have only one conjugate molecule, and besilver enhanced to form a PRP with that conjugate molecule attached onthe outermost surface thereof. A 20 nm nucleation center will have acorrespondingly larger number of conjugates attached, some of which willend up bound to the surface of the silver enhanced PRP. During silverenhancement, the conjugate bound PRPs can be incubated with a blockingagent such as bovine serum albumin (BSA) to reduce the presence ofnon-specific bonding sites on the surface of the PRP.

In another embodiment, a conjugate is added to a PRP after the PRP ismade. The conjugate associates with the PRP either covalently ornoncovalently. For example, a fully formed PRP is coated with gelatin,agar, polytetrafluoroethylene (Teflon™), PVP, or latex to preventnon-specific charge interactions, followed by covalent attachment of oneor more functional groups thereto by well known methods, thus generatinga PRP attached to a first half of a particular conjugate pair. Thereagents required to couple conjugates to immunogold nucleating centersor formed PRPs are all commercially available.

This method of forming conjugate bound PRPs also allows control of thenumber of conjugate molecules (i.e. “first half” conjugate pairs) boundto the surface of the PRE. In this case, one can incubate bare PRPs in asolution of conjugate and a blocking agent such as BSA. The relativeconcentrations of conjugate and BSA can be adjusted to produce, onaverage, the desired amount of conjugate on each PRP. For PRPs ofunusual shape, or comprising concentric shells of different materials,coating with conjugates after formation is typically more convenient.Conjugate bound PRPs which are approximately spherical, however, canconveniently be produced from commercially available conjugate boundgold colloid nucleating centers and silver enhancers as described above.

Conjugates or other molecules may be bound directly to the metal surfaceof the PRP. The surface chemistry involved in such binding is complex,but it is currently exploited extensively in many non-PRP immuno-goldsilver staining techniques. Alternatively, the PRPs may be coated with ashell of plastic material such as latex prior to the binding ofadditional molecules such as conjugate. Techniques for binding moleculesto latex are also well known. The molecules bound to the metal directlyor plastic shell may be the conjugate itself, or may be otherintermediate reactive groups such as sulfides, amides, phosphates,aldehydes, carboxyl, alcohol, or others to which conjugate or othermolecules of interest may be bound. Conjugates or other molecules ofinterest may be synthesized onto such a reactive base with knowntechniques of combinatorial chemistry.

E. Formation of PRP Populations With Desired Characteristics

The differences in emission spectra for the two separate PRPs as shownin FIG. 1 can arise from a number of factors. One significant factor issize, particles of larger size having resonance peaks at longerwavelengths, and also having spectral shapes with increased half-maximumwidths. Therefore, control of the size of PRPs being produced results incontrol over some spectral characteristics.

It can thus be appreciated that with the addition of a controlled amountof enhancer, a population of PRPs with a narrow range of diameters, andtherefore a correspondingly narrow range of resonant peak frequencies,may be produced, such as is illustrated in FIG. 2 for four types ofPRPs. In some advantageous PRP production methods, the particles can beobserved during the enhancement process with a suitable microscope.These methods use enhancement chemicals such as are described hereinwhich are relatively unaffected by incident light needed to observedevelopment of the PRP during the enhancement process. Thus, PRPdevelopment can be observed and halted when it has reached a desiredend-point. For those applications in which it may be desirable to use alight sensitive enhancement process, or if development outside themicroscope is desired, timed sequential enhancement is performed. Thesamples are rinsed after each application and the status of PRP lightscattering is determined. One can continue with as many sequentialenhancement steps as desired.

As indicated above, the PRP composition of the invention includes one ofmore PRP populations having peak wavelengths 40 nm of a definedwavelength. Such homogeneity in PRP population is possible by stepwiseaddition of enhancer coupled with darkfield observation of the PRPcreation as described above. Because the width of a plasmon resonancepeak is typically 20 to 40 nm, it is generally unnecessary to furtherreduce the variance in resonance peaks of the PRP population.

Populations of PRPs having uniform spectral characteristics canalternatively also be prepared by purifying non-homogeneous populations.PRP conjugates and free PRPs can be separated by conventionalbiochemical methods including column chromatography, centrifugation,electrophoresis and filtration. Because PRPs with surface localizedmolecules or entities can have a significantly different mobility thando free PRPs of the same size, they elute from gel filtration columns ata different rate than do free PRPs. Because PRPs are charged particles,they migrate in an electric field. Thus, PRPs can be manipulated by andeven observed during electrophoresis.

PRPs having certain desired characteristics can also be separated basedon their Zeta potentials. Zeta potential separation equipment suitablefor this use is commercially available (Coulter Corp, Florida).Radiation pressure may also be used to force PRPs through a matrix atdifferent rates depending on their structural properties. If bound andfree PRPs are subjected to electrophoresis in, for example, an agaroseor acrylamide gel, the free PRPs migrate faster than do the bound PRPs.Likewise, PRP conjugates may be preferentially retained by filters.Purification can alternatively be performed by centrifugation. Thus,with these methods, an original population of PRPs having a wide rangeof spectral characteristics can be separated into subpopulations whichhave a narrower range of spectral characteristics.

Individual populations of PRPs, may be prepared separately and latermixed according to a desired combination of PRP property, e.g., color,in desired amounts, each labeled with the same or different biologicalmacromolecules or unlabeled depending on the application. In suchcompositions, it is preferable for the resonance peaks of the differentpopulations of PRPs to be substantially non-overlapping, as definedabove. In some preferred embodiments, the variance in peak location ofone population of PRPs is controlled to be within approximately 20 nm ofone defined wavelength, and the variance in peak location of anotherpopulation of PRPs in the mixture is controlled to be withinapproximately 20 nm of a second defined wavelength. To avoid significantoverlap, it is preferable to ensure that the two peak wavelengths are atleast 30 to 40 nm apart, and most preferably 50

or more nanometers apart.

Applying the above methods, new types of molecular probes can beproduced by binding selected conjugates to selected PRPs. As mentionedabove, a PRP population with a first spectral characteristic can bebound to a first conjugate, and a PRP population with a second spectralcharacteristic can be bound to a second, different conjugate to producetwo differentiable populations of PRPs with different preferentialbinding properties. Such pre-defined mixtures of PRPs are especiallyuseful in improving the accuracy of detection of low abundance moleculesas will be explained further below.

F. Isolated Non-Spherical and Composite PRPs

The emission spectra of PRPs is further affected by the details of theirstructure. Ellipsoidal PRPs offer additional parameters foridentification and discrimination. Each ellipsoidal PRP may have two orthree plasmon resonant peaks, depending on whether there is one isotropyaxis or three different principal axis dimensions, respectively.Ellipsoidal PRPs having one isotropy axis show peaks corresponding totwo orthogonally polarized emissions, one associated with plasmonexcitation along the major axis of the ellipse, the other associatedwith plasmon excitation along the minor axis. The distinct plasmonresonance peaks occur at maximal intensity when the polarization ofincident light is along the corresponding principle axis. Thus, theresponse of a fixed ellipsoidal PRP to polarized light may vary with thedirection of incidence.

A process for making ellipsoidal silver particles consists of pulling ona glass matrix containing spherical PRPs at a temperature such that theviscosity results in a stretching of the PRPs into prolate ellipsoidalparticles having a desired aspect ratio. Conditions have also beendescribed for “pushing” on such particles such that they form oblateellipsoidal particles. One ellipsoidal PRP containing matrix, Polarcor™(Corning Company, Corning, N.Y.), consists of aligned ellipsoidal PRPsin a glass matrix. This composite material is an effective polarizer forcertain optical frequencies, principally in the red and above.Individual ellipsoidal PRPs contained within such a glass matrix can beisolated by dissolving the matrix in such a manner so as to not disturbthe PRPs. By preparing ellipsoidal gold particles of the correct aspectratio and size in a suitable matrix, then dissolving the matrix in amanner that does not disturb the particles, large quantities ofellipsoidal of PRPs having approximately equivalent plasmon resonancescattering properties can be prepared for use in, for example, liquidreagent preparations for biochemical assays as described in detailbelow.

Other methods can be used to prepare ellipsoidal PRPs, which may beproduced via photoreaction of silver halides or with appropriatelithographic molds. Alternatively, PRPs which are originally formed tobe substantially spherical can be pressed or rolled between two surfacesto flatten them into a desired oblate or prolate ellipsoidalconfiguration.

Non-spherical and non-ellipsoidal PRPs are observed to havecharacteristic spectral signatures which are different than sphericaland elliptical particles, and which are useful in creating plasmonresonant probes with advantageous size and scattering properties. It hasbeen found that PRPs which scatter strongly in the blue region may beconveniently produced by making spherical silver particles. As discussedabove, increases in particle diameter will red shift the resonant peak.However, for spherical particles, the peak intensity of the scatteredlight begins to drop off as the peak is shifted into the red, andaccordingly strong red scatterers are much more difficult to producewith spherical particles than are strong blue scatterers. Particles ofother geometric shape, however, can produce strong scattering at longerwavelengths. Three such particles are triangular, square, andapproximately hexagonal in cross-section. Triangular, square, andhexagonal silver particles may, for example, be produced viaphotoreaction of silver halides or with appropriate lithographic molds.Isolated hexagonal particles of a similar size as a blue sphericalparticle will typically have a green plasmon resonance peak. Theisolated triangular particles, which may have 50-150 nm characteristicdimension, are of particular interest because they often exhibit aresonance peak in the red part of the visible spectrum. It has beenfound that production of triangular PRPs is one suitable method ofobtaining PRPs which appear red. A specific example of a red triangularparticle and a blue spherical particle are discussed below withreference to FIGS. 7 and 8. It is also possible to bind small sphericalmetal particles into pairs or other conglomerates to form a variety ofplasmon resonant particle shapes.

In addition, PRPs having concentric shells of dielectric and conductivematerial can be prepared. In these embodiments of PRPs, the peak of theplasmon resonance can be tuned to a desired frequency. Specifically,PRPs can be made with the addition of dielectric material as either thecore or external shell tends to red shift the resonant peak and producesa comparably strong scatterer. For this reason, red PRPs mayadvantageously be produced with the inclusion of such a dielectric shellor core. Particles having a dielectric core and a shell of aluminum havebeen found to have a plasmon resonance peak in the ultra-violet, atapproximately 240-280 nm.

Particles of three layers comprising a dielectric core/metalshell/outside dielectric shell may be useful for further flexibility inchanging the peak response and the scattering strength. In addition, aPRP with multiple concentric conductive shells can be created. Becauseeach shell will have a different diameter, complex scattering spectraoften containing several separate peaks can be produced. These peaks canbe shifted with variations in the dielectric material separating theconductive shells. As will be explained in more detail below, adielectric outer shell, comprising latex, teflon, or other polymercoating, is also useful as a substrate suitable for bindingmacromolecules of interest to the outside of the PRP.

The production of PRPs having multiple shells of conductive material anddielectric can be more complex, but these may be manufactured withvarious film deposition techniques including chemical vapor depositionor sputtering. Other methods for fabricating multi-shelled PRPembodiments are described in U.S. Pat. No. 5,023,139 to Birnboim et al.,mentioned above.

A PRP having a dielectric core and an outer metal shell can also be madewith electroless plating techniques. In this process, core particles,made, for example, from latex, have their surfaces activated with metalatoms which may be platinum atoms. Using enhancement procedures asdescribed above, these platinum atoms comprise nucleation centers forsilver enhancement and the formation of a shell around the latex core.

A PRP compositions which may, but do not necessarily include all of thelimitations (i)-(iv) in the PRP composition just described are alsocontemplated herein, as novel PRP compositions for use in a variety ofapplications discussed herein, including the general method disclosed inSection III.

Two-ligand composition. The composition contains two populations ofPRPs, each having a different ligand species carried on the PRP surface.The two ligands are designed to bind to different ligand-binding siteson a target. The two populations PRPs may have different spectralproperties.

Fluorescent-reporter composition. The composition includes PRPs havingsurface attached ligands, for binding to the ligand-binding sites of atarget. The composition is a very sensitive, “onesite” reporter, in thatfluorescence emission excited by the plasmon resonance spectral emissionof the associated particle acts to focus excitation light at the site ofthe fluorescent molecules. The composition may also be sensitive to thetarget environment, if such is designed to contain fluorescencequenching or fluorescence energy transfer molecules.

Fluorescent Quenching or Energy Transfer. This composition includes twopopulations of PRPs, each having a surface-attached ligand (which may bethe same or different) for binding to two proximate sites of a target.Each population contains surface-localized florescent molecules whicheither produce fluorescence quenching when proximately disposed, orwhich contain donor and acceptor fluorescent molecules for non-radiativeenergy transfer when proximately disposed. The composition is useful,for example, in a homogeneous assay for detecting a target with firstand second proximate ligand binding sites.

PRPs with Raman-active entities. The composition includes a plurality ofPRP populations, each with a different Raman-active entity localized onthe PRP surfaces. Each population may contain additionalsurface-localized molecules, e.g., oligonucleotides with different basesequences or combinatorial library molecules, where the identity of eachsurface-localized molecule is associated with a Raman-active entity,e.g., molecule, with a known, unique Raman spectrum signature. Thecomposition is used, for example, to identify combinatorial librarycompounds that are (i) formed on the PRPs according to standardbead-synthesis methods, and (ii) identified as having a desired compoundactivity.

As another example, the composition is used for chromosome mapping,where the relative spatial positions of known sequence regions, e.g.,ESTs or SSTs, are determined by (i) attaching to each PRP with a uniqueRaman spectral signature, an oligo sequence fragment complementary toone of the chromosome sequences, (ii) hybridizing the probes with thechromosomal DNA, and (iii) identifying from the unique spectralsignature of each PRP, the relative position of the PRPs bound to theDNA. By placing the DNA in an extended condition, as above, the mappingdistances separating the sequences can also be determined. FIG. 13 showsthe binding of an DNA-sequence labeled PRE to a Drosophila polytenechromosomes, illustrating the ability to localize PREs in a chromosomeregion.

V. Diagnostic Methods and Compositions

The diagnostic method of the invention is intended for use indetermining the presence of, or information about, a target having amolecular feature of interest. The method is preferably practiced inaccordance with the method and apparatus described in Section III, andpreferably employing the PRPs described in Section IV, where the PRPshave surface localized ligands.

In practicing the method, the target is contacted with one or more PREshaving surface localized molecules, to produce an interaction betweenthe molecular feature and the localized molecules. This interaction mayinclude (A) binding of a PRE to a target binding site, for example,through a ligand/ligand-binding interaction, to produce a PRE/targetcomplex, (B) binding of two PRPs to closely spaced target sites, toproduce a spectral characteristic evidencing a PRE/PRE interaction, (C)cleavage of a linkage between two PREs, to produce unlinked PREs, (D)binding of a PRE to a target, e.g., through a ligand/ligand-bindinginteraction, to alter the Raman spectrum of Raman-active molecules on aPRE in a detectable fashion, (E) binding of a PRE to a target, e.g.,through a ligand/ligand-binding interaction, to alter, e.g., quench orenhance the intensity of the fluorescence emission of fluorescencemolecules on a PRE in a detectable fashion, and (F) formation of alinkage between PREs to produce coupled PREs.

The target is illuminated with an optical light source, in a mannerwhich allows one or more selected plasmon resonance spectral emissioncharacteristics to be determined, as detailed in Section III. Thepresence of or information about the target by is then determined bydetecting a plasmon resonance spectral emission characteristic of one ormore PREs after such interaction with the target.

The PREs employed in the method are preferably PRPs constructed as aboveto contain a surface-localized molecule that is one of aligand/ligand-binding site conjugate pair, such as antigen/antibody,hormone/receptor, drug/receptor, effector/receptor, enzyme/substrate,lipid/lipid binding agent and complementary nucleic acids strands. Wherethe spectral emission characteristic detected is related to a shift orchange in Raman or fluorescence spectral characteristic, the PRPs alsocontain surface-localized fluorescent or Raman-active molecules ofentities, respectively. The PRPs employed may have the quality andhomogeneity attributes of the PRP composition disclosed in Section IV,or may have less stringent uniformity attributes.

Because PRE probes are extremely sensitive (as noted above, one canobserve and spectrally analyze a single PRE), the method may be madevery sensitive to amount of target analyte (down to one event). Thisallows earlier detection of pathogens in bodily fluids, includingearlier detection of HIV, tumor markers and bacterial pathogens,detection from smaller fluid volumes, and the ability to miniaturizemany existing diagnostic tests.

A. Binding of a PRE to a Target Binding Site

In this general embodiment, PREs with surface ligand molecules arecontacted with a target under conditions that lead to PRP-bound ligandbinding to ligand-binding sites on the target, forming one or morePRE/target complexes with the target. Typically, the spectral emissioncharacteristic(s) being measured are unchanged by complex formation.That is, neither PRP/PRP proximity spectral emission effects or changesin spectral emission characteristics caused by PRP interactions with thetarget are observed.

Typically in this embodiment, the target being analyzed is immobilizedor competes for an immobilized binding site. After PRP binding to thesolid phase, immobilized surface, the solid phase is washed to removenon-bound PRPs before illuminating the target and detecting a plasmonresonance spectral characteristic of the target complex(es). The PREscontacted with the target may include two or more populations, each withdifferent ligands, and preferably each with different spectralsignatures associated with different ligands, e.g., blue particles forone ligand, and red particles for another. As will be detailed below,this embodiment has applications for:

(i) detecting the presence of an target analyte, where the analyte iseither immobilized, competes with an immobilized binding agent, or canbe separated from unbound PRPs in the contacting mixture;

(ii) in situ hybridization of PRP-oligonucleotide conjugates with a DNAtarget, to isolate PRPs at the site of sequence hybridization;

(iii) mapping spatial features of the target, for example, thearrangement of a specific binding site on a target cell or tissue, or ina DNA target, for chromosome mapping; and

(iv) In situ labeling of a target, for example, in a Southern blot,directly binding probe-labeled PRPs to a DNA fractionation gel, toidentify separated DNA bands.

The following examples illustrate various assays in which PRPs withsurface attached ligand molecules are bound to immobilized targettissue, for purposes of detecting spatial features of the binding sites,and/or the density of binding sites.

EXAMPLE 4 Labeling of Ryanodine Receptor in Chicken Muscle with PREs

Frozen chick intercostal muscle fixed in paraformaldehyde was cut in 2-3μm sections and transferred to prepared coverslips (Cell Tak coatedspots in Pap pen wells). Tissue sections were washed three times for5-10 minutes each time with PBS. Nonspecific binding sites were blockedby incubation in 3% normal goat serum, 1% gelatin, 0.01% Triton X-100 inPBS for 20 min. Coverslips were washed for 5 min with a 1:3 dilution ofblocking buffer (working buffer), then incubated in a 1:5 dilution ofmouse anti-ryanodine monoclonal antibody (34C) in working buffer for onehour. Coverslips were then washed 6 times for 3-5 minutes each time withworking buffer, followed by incubation with a 1:40 dilution of 5 or 10nm gold particles conjugated to goat anti-mouse IgG (AuroProbe EM,Amersham) for 30 minutes. Coverslips were washed 3 times for 3-14 5minutes each with working buffer, then 3 times for 3-5 min with PBS.Samples were washed 3 times for 2 min in doubly distilled water, thensilver enhanced for 8 min using 50 μl initiator, 50 μl enhancer (IntenSEM Silver Enhancement kit, Amersham) and 1 mil 0.1 mg/mil gelatin.Samples were washed three times for 3-5 min with doubly distilled water,covered with Gelvatol anti-fade media and visualized under darkfieldmicroscopy. Individual PREs were observed regularly spaced along theZ-lines of the muscle which contain the ryanodine receptors. The resultsare shown in FIG. 12.

EXAMPLE 5 Binding of PREs to DNA on Nitrocellulose

A 1 cm×3 cm piece of nitrocellulose membrane was cut and the top surfacewas marked at one end with a pencil to identify the DNA surface.Biotinylated DNA (100 ng) was pipetted onto one end of the surface ofthe marked nitrocellulose. Non-biotinylated DNA (100 ng) DNA was appliedto the other end as a control. The nitrocellulose was placed in adesiccator to dry the DNA spots, then cross-linked with ultravioletlight. The nitrocellulose, DNA surface up, was placed in a smallparafilm dish and incubated for at least 4 hours in blocking solution(50 mM sodium phosphate, 150 mM NaCl, 2% BSA, 1% Tween-20) at roomtemperature in a 100% humidity chamber to prevent evaporation. A goatanti-biotin immunogold conjugate (Nanoprobes, Inc., Stony Brook, N.Y.;about 1×10⁸ colloids), was added and the incubation continued for 2hours at room temperature. Immunogold conjugates were removed, blockingsolution (500 μl) was added, and the nitrocellulose was washed for onehour at room temperature. Blocking solution was removed and the filterthoroughly rinsed with doubly distilled water. Double distilled water (2ml) was added to the filter which was washed for 30 min. After removalof the water, one ml enhancer solution (1 ml 0.1 mg/ml gelatin, 50 μlinitiator, 50 μl enhancer; enhancer and initiator were from BBIInternational, United Kingdom, Ted Pella Catalog #SEKL15) was placed onthe nitrocellulose for 8 minutes. The enhancer solution was then washedaway thoroughly with distilled water. The nitrocellulose was placed onfilter paper, DNA side up, in a desiccator to dry for one hour, thenplaced on a glass slide, DNA side up, and covered with a glasscoverslip. The slide was placed in an acetone fume chamber for 20minutes or until the nitrocellulose became transparent. PREs were viewedusing a darkfield microscope. A high density of PREs were observed wherebiotinylated DNA was spotted compared to a low density of PREs elsewhereon the nitrocellulose, including where non-biotinylated DNA was spotted.The same experiment also successfully detected biotinylated DNA whenfully formed PREs conjugated to goat anti-biotin were used.

EXAMPLE 6 Binding of PREs to DNA in Polystyrene Cell Culture Dish

Doubly distilled water (150 μl) and 1 M NaHCO₃ (17 μl) were added to onerow of wells in a 48 well culture dish. Neutravidin (10 μg) was added toeach dish followed by incubation overnight. All incubations wereperformed at 4° C., 100% relative humidity. Neutravidin was removed andthe wells were thoroughly rinsed with doubly distilled water. 150 μl ofblocking buffer (50 mM sodium phosphate, 150 mM NaCl, 2% BSA, 1%Tween-20) was added to each well followed by a 4 hour incubation.Beginning with the second well, 1 μg biotinylated DNA was added to theblocking buffer. In subsequent wells, 10-fold dilutions of biotinylatedDNA were added (0.1 μg, 0.01 μg, . . . ) and the plate was incubated for4 hours. Biotinylated DNA was removed and the wells were thoroughlyrinsed with doubly distilled water. Blocking buffer (150 μl of thebuffer described above) and approximately 10⁸ goat anti-biotinconjugated immunogold colloids was added to each well and incubated for4 hours. The immunogold colloid was then removed, wells were thoroughlyrinsed with doubly distilled water, and colloids were enhanced byapplying 100 μl enhancer solution to each well for 7 minutes. Enhancersolution was removed, wells were thoroughly rinsed with doubly distilledwater and wells were dried with dust-free compressed air. PREconcentration in each well was determined using darkfield microscopy.The PRE concentration was correlated with the DNA concentration in eachwell. The same experiment also successfully detected biotinylated DNAwhen fully formed PREs conjugated to goat anti-biotin were used.

Fluorescent in situ hybridization (FISH) may be performed with PREs(PRISH) instead of fluorescent labels. In this method, a PRE-labeledoligonucleotide is incubated with a DNA molecule of interest. Ifcomplementary sequences exist on the PRE bound oligonucleotides, thebound PREs may be observed. Alternatively, two PREs, each attached to adifferent oligonucleotide, are incubated with a DNA molecule ofinterest. If a genetic deletion associated with a particular disorder ispresent and the PREs bind on either side of the deleted region, theywill be much closer together in the deletion versus the wild type. Inthis method, bound pairs of PREs may be detected by alterations inscattering parameters, or by observing correlated pairs of PREs ofeither the same or different spectral characteristics. PRISH allowsdetection of a smaller defect or selected genomic region due to gains inlocalization. With appropriately conjugated PREs, bound PREs may beobserved at several locations along a strand of nucleic acid, providinginformation about several sites at one time. Distance measurements canalso be made, as discussed in detail above. As illustrated by FIGS. 7and 8, a 230 nm distance between PREs, which corresponds toapproximately 640 base pairs, can be easily resolved to an accuracy ofonly tens of nanometers or less with optical microscopy. In addition,the use of PCR or other enhancement steps is unnecessary in contrast toFISH in which enhancement is usually required, although PCR enhancementcan also be used in conjunction with PRE hybridization tests. Geneticdeletions and mutations can also be detected using a ligase to join twoadjacent strands of PRE coupled nucleic acid. If the PRE coupled strandshybridize in a precisely adjacent manner, denaturing will result in freestrands of nucleic acid coupled to a pair of PREs. These bound pairs maythen be observed as described above. If the strands hybridize atlocations which are too close or too far apart, the ligase reaction willnot occur, and bound pairs will not form. Bound pairs of PREs may alsobe produced with PCR methods if PREs are coupled to hybridizing strandsof nucleic acid, and standard PCR techniques are used to amplify thequantity of target nucleic acid present.

In all of these procedures, the PREs can be conjugated to theoligonucleotides either before or after the oligonucleotides are boundto the target nucleic acid. Furthermore, such tests can be performed invitro, or in a cell. Selective PRE hybridization is also advantageouslyapplied to screening multiple nucleic acid containing sample wellscomprising a library of different nucleic acid sequences. Such samplewells may be provided on library chip arrays or standard multiwelldishes.

PREs can also be coated with antibodies for use in assays analogous toenzyme-linked immunosorbent assay (ELISA) detection of variousmacromolecules. In one advantageous embodiment, multi-well dishes (i.e.96-well microtiter plates) are coated with an antibody specific for amolecule of interest. A biological fluid to be tested is then placed inthe wells containing the immobilized antibody. A PRE-labeled secondaryantibody which binds a different region of the molecule than does theimmobilized antibody is added to the wells. The plate is then read witha plate reader compatible with darkfield optical detection. The presenceand level of PRE binding indicates the presence and amount of moleculein the biological fluid.

In another embodiment, a particular sample can also be visualized usingmultiple populations of PREs, each having a distinct spectral signature,and conjugated to separate antibodies which recognize different bindingsites on a target molecule, or which recognize different targetmolecules. Alternatively, the PREs are coupled to a polyclonal antibodywhich recognizes a plurality of epitopes on the same target protein. Thepresence of two spectrally distinct PREs at the same location indicatesa positive signal, while the separate presence of either particle wouldconstitute an incomplete identification and would be rejected. Thisapproach significantly reduces false positive signals in clinicaldiagnostic assays.

Additional advantages of PRE immunoassays include the fact that theability to detect one PRE with a good signal to noise ratio obviates theneed to amplify the signal by using secondary antibodies or enzymes andtheir substrates. This further eliminates non-specific background.Moreover, the ability to analyze the sample during processing by opticalmicroscopy allows real time correction of incubation and wash conditionsso as to further optimize signal to noise. PRE assays may beconveniently employed with essentially any target substance and variousbinding partners in direct, sandwich, and other widely used testformats, some of which are described in more detail below. Substancestested for and conjugated to PREs include proteins, nucleic acid,ligands, receptors, antigens, sugars, lectins, enzymes, etc.

EXAMPLE 7 PRP Assay of Goat-Antibiotin

The wells of a polystyrene multi-well dish were coated with biotinylatedBSA. Regular BSA was added to block any remaining non-specific bindingsites in the wells. Samples of goat-antibiotin antibodies ranging inconcentration from 0.06 to 600 picograms (pg) were added to individualwells. A control sample having no goat-antibiotin antibodies was alsoassayed. PRPs bound to rabbit-antigoat antibodies were then added toeach well and incubated. Unbound PRPs were washed from the wells, andbound PRPs in each well were observed with a darkfield opticalmicroscope. Light sources in the field of view were analyzed accordingto the discrimination techniques described above, and the remainingscattering sites were individually counted in each well. The results ofthis test are shown in FIG. 9. The control sample had one countremaining after image processing, and is illustrated as the dark bar inFIG. 9. The number of counted PREs over the concentration range testedvaried from 4 at 0.06 pg analyte, to over 1000 at 600 pg analyte.

Because it is advantageous to perform these assays with one or morepopulations of PREs having approximately uniform spectralcharacteristics, it is advantageous to form the PRE labels first underconditions which are conducive to forming such approximately uniformpopulations. As mentioned above, the binding of nucleation centers tobinding sites, followed by metal enhancement and optical observation hasbeen described, but this technique provides very little control over thespectral characteristics of the particles thus created. And even whenthese techniques have been performed, no effort to use the PREscattering characteristics to discriminate background and make a highlysensitive assay has been made or proposed. Accordingly, PRE assaysperformed by first labeling target species with nucleation centers, andthen metal enhancing them to form PREs, (rather than forming the PREsprior to binding) are still improved when light discrimination betweenPRE scatterers and background is performed. Furthermore, assays whichuse pre-formed optically observable sub-wavelength light emitters of anykind have not taken advantage of the technique of individually countingparticles to create a sensitive assay. When spectral and spatialdiscrimination of background is performed, such counting can be usefulfor fluorescent or luminescent bead labels in addition to PRE labels. Aswill be discussed below, fluorescence can be enhanced by local plasmonresonance, and thus PRE enhanced fluorescent beads provide an additionalsub-wavelength light emitting label useful for such assays.

It can also be appreciated that many variations of these types of assaysmay be performed with PRE labels. All of the various types ofimmunosorbent assays which are currently performed using fluorescentmolecule labels may be performed with PREs instead. Sandwich andcompetition assays, for example, may be performed with PREs. In thefirst case, an entity such as an antibody having affinity for a targetsubstance to be detected may be immobilized on the bottom of an assaywell. A test sample including the target substance is added to the well,and the target substance binds to the first entity. A second entity,having affinity for a different portion of the target substance, maythen be added to the well, wherein it binds to the target species.Finally, PREs bound to a third entity having affinity for the secondentity are added to the well, which bind in turn to the second entity.After rinsing, it can be appreciated that PREs will only be bound tosites where the target substance has been previously bound. This test isvery useful when the first and second entities mentioned above areantibodies having affinity to different epitopes on an antigen beingassayed. The third entity, bound to the PREs, may then be ananti-species antibody, rather than being a specific binding partner ofthe target substance.

In a competition assay, a first entity may be immobilized in an assaywell, and both PRE coupled second entities and target substances areadded to the well, wherein the second entity and the target substancecompete for binding to the first entity. When unbound PREs areseparated, the number of PREs remaining in the well indicates the extentto which the target substance was able to occupy binding sites. In thistype of assay, the PRE bound second entity may be the same as the targetsubstance, or may be a different substance which also has an affinityfor the first entity.

Those of skill in the art will recognize that PRE labels may be used tobind to a wide variety of molecular complexes in a wide variety of waysto produce a sensitive assay. As additional examples, the conjugate onthe PRE label may be a specific binding partner of the analyte beingtested for. It may be a specific binding partner of an immobilizedanalyte/antibody complex. As another alternative, PRE may bind to animmobilized antibody, but only if that immobilized antibody haspreviously bound an analyte molecule. Each of these various techniquesmay be especially suitable in a given assay, depending on the chemicalnature of the analyte being tested for.

Furthermore, it will be appreciated that assays for multiple analytescan be performed simultaneously using populations of PREs havingdifferent spectral signatures. Populations of PREs different color ordifferent polarization responses can be conjugated so as to recognizedifferent target substances. When introduced into a matrix containingunknown concentrations of several different analytes, all of the assaysset forth herein could be performed on several target substances at onceby separately counting the PREs associated with each distinctivespectral characteristic.

PRE probes can also be used to screen in vitro combinatorial libraries.In some conventional versions of this technique, a drug receptor islabeled with a fluorophore then mixed with beads, the collection ofwhich constitutes the combinatorial library, and spread out on a slide.The presence of a fluorescent bead indicates receptor binding and thepresence of a potential drug bound to the bead. In one embodiment of theinvention, the fluorescent receptor is replaced with a PRE-labeledreceptor which increases the sensitivity and photostability of theassay, thereby allowing for the possible production of the originalcombinatorial library on smaller beads and the ability to synthesize andscreen larger chemical libraries.

The libraries may also be synthesized on microchips, where the presenceof a PRE probe indicates receptor binding. Recent applications ofcombinatorial libraries for improved drug discovery may thus be enhancedby using PRE probes as a method of detection of potential candidates.Selectively attached PRE increase the resolution and sensitivity ofbio-chip detection schemes.

In all of these assays, PRE calibration is conveniently performed usingPREs of different spectral characteristics than are used to detect thetarget entities. In essence, the assays are calibrated by introducing apredetermined quantity of PREs having a selected spectral characteristicto create a control population of PREs which can be detected andmeasured in conjunction with the PREs used for the assay function. Asone specific sandwich assay calibration example, red PREs may beconjugated to the target entity being tested for, and a known amount(but of course much lower than a saturating amount) of this PREconjugated target entity is added to the well along with the sample,either sequentially or simultaneously. After rinsing away unboundconjugated red PREs, antibodies to the target entity are added. Afterrinsing unbound target entity antibodies, blue PREs (for example) whichare conjugated to an anti-species antibody are added which bind to theantibody to the target entity. After rinsing, both red and blue PREs arecounted, and the red PRE count provides a calibration count. In analternative to this sandwich format, a direct binding assay calibrationmay also be utilized, wherein different immobilized antibodies areprovided on the bottom of the sample well, and the calibration PREs areconjugated to a specific binding partner to one of the immobilizedantibodies.

Assays with PREs can also be performed in cells. Conjugated PREs can bebound to both fixed or free sites in cells and their locationsindividually observed. Well known techniques exist for placement ofparticles into cells, including high pressure bursts which cause theparticles to perforate the cell membrane and electro-perforation inwhich high voltage discharges are used for the acceleration process (thePRE is typically charged prior to the electro-perforation techniques).Apparatus for performing these techniques are available from BioRadLaboratories of Hercules, Calif. PREs and PRE conjugates may also beintroduced into cells by conventional transfection techniques includingelectroporation. PREs can also be placed into cells directly by piercinga cell membrane with a micropipette, and directly injecting one or morePREs into the cell. In a preferred embodiment, the PRE is coated (i.e.latex) by well known methods to protect it from biochemical damage.

In some advantageous embodiments of PRE assays within living cells, twopopulations of differently conjugated PREs are inserted into one or morecells. The separate conjugates associated with each separate populationmay be selected to bind to a different epitope on a target substancebeing manufactured in the cell. After injection into the cell, presenceof the target substance will be indicated by PRE pairing, which isdetected using the techniques described above. Depending on the natureof the target substance, it may be desirable to have PREs with similar,or disparate spectral characteristics associated with each conjugate.

It is advantageous to prepare wells for use with PRE assays which aresuitable for observation with darkfield microscopy. For the multi-wellplates to include a substrate suitable for darkfield microscopy, thewell bottoms are advantageously manufactured with particular emphasis onuniformity, smoothness, and cleanliness so as to hinder the formation oflight scattering imperfections. Such care is currently not taken in theproduction of standard 96 well dishes. In addition, the outside surfaceunder the wells should also be relatively clean and smooth, as theoutside surface also provides a light scattering surface which canintroduce undesired background signals. In some advantageousembodiments, the surfaces of the wells have less than approximately 100,or even less than approximately 10, light scattering imperfectionstherein. As an additional method of increasing signal to noise ratios inthese assays, the location of imperfections in a well can be documented,and a scattering signal from those locations can be ignored when theassay is performed with that particular well.

Typically, the field of view of the optical microscopes used in theseassays comprises all of or portions of the bottom of the well. Thus,when low levels of analyte are being detected, it can be important toensure that a minimum amount of analyte stick to the walls of the well,rather than to the bottom. It is accordingly advantageous to include ablocking agent on the walls of the well during production. To make sucha well, a dish may be inverted and placed on a solution including ablocking agent such as BSA. If the dish is pushed down into thesolution, or some of the air trapped in the wells is removed by suckingit out with a pipette or capillary, the BSA solution can be made tocontact the walls of the well without touching the bottom of the well.After this step, the desired antibody or other binding agent isimmobilized on the bottom of the well, and then additional blockingagent may be added to block remaining nonspecific binding sites on thewell bottom.

Assay methods according to the invention can also be automated,employing, for example, the method and apparatus of the inventiondescribed in Section III. Automated plate readers are currently used forconventional assay techniques, and the principles for a robotic PREplate reader are in some ways similar. As with currently available platereaders, a robotic sample loader may or may not be provided. A roboticPRE assay plate reader would advantageously include sample wells and amicroscope for observing all or portions of the bottom of the wells. Insome embodiments, a very small objective lens, which may beapproximately 2 mm in diameter, is lowered down into the well and closeto the well bottom to obtain a high numerical aperture while imaging aportion of the well bottom. In these embodiments, the PREs may beilluminated from the bottom with total internal reflection off thebottom of a transparent well bottom. As the light is gathered by theobjective, light emitting entities can be detected and discriminatedfrom background using automated image analysis techniques as describedabove. Counting the remaining discriminated particle sources can also beautomatically performed. In some embodiments, the field of view of themicroscope may be a portion of the assay well bottom, and the reader maybe configured to discriminate and count particles in several regions ofthe same assay well until a certain predetermined count is read. Onlyafter this count is reached will the reader move to a different assaywell. This technique will save time by moving quickly from well to wellwhen a large signal is present, and will take the time required toobtain adequate count statistics when low numbers of bound PREs arepresent in the well. The reader may also be configured to performadditional levels of discrimination depending on the count received. Forexample, a first discrimination based on the spatial deviation from theexpected point spread function may be performed for all fields of view,but an additional spectral deviation measurement will be made when a lowcount value is obtained. All of the thresholds for performing variouslevels of discrimination can be preprogrammed into the reader, againinsuring that wells having low PRE counts are analyzed to maximizesignal to noise ratios, while time is saved on wells having a largenumber of counts, where signal levels are already high.

It will also be appreciated that mercantile kits including ingredientsfor performing assays described herein may be created having novelcombinations of ingredients. Advantageously, such kits may include acontainer of PREs having approximately equal spectral characteristics.The PREs may be conjugated to selected biological molecules such asantibodies or other types of specific binding partners for selectedsubstances. They may be coupled to reactive groups for custom formationof conjugate at a later time. Washing and blocking solutions may also beprovided. A second container of PREs may also be provided forcalibration or multiple assays as described above.

B. Binding of two PRPs to Closely Spaced Target Sites

As discussed above, the spectral characteristics of light emitted byPREs is dependent on their proximity to other PREs. Changes in observedpeaks in emitted frequencies, e.g., peak wavelength, spectral width athalf intensity, the appearance of more than one peak, and changes inresponse to polarized light, etc., can all be observed as PREs approachand move away from one another. These features can even be used todetermine the approximate distance between PREs, by measuring the extentof their interaction.

Agglutination and aggregation-dependent immunoassays are thus performedusing PRE probes, and have the capability of single molecule detection.In one embodiment, two antibodies are each attached to a PRE probehaving either the same or distinct spectral signatures. These antibodiesbind to the same biomolecule of interest, but at non-competitive sites.The distance between the two binding sites will place the PRE probes inclose proximity which are directly detected via narrow band illuminationif the two PRE have separated plasmon resonance frequencies or if theyhave the same plasmon resonance frequency, by a unique spectralsignature as a result of their interaction. For example, blood serum isadded to a tube containing PRE probes which have been coated withantibodies specific for a particular serum component. After incubation,the sample is spread on a glass slide and the frequency of aggregated(i.e. close proximity) PRE probes is determined. This is compared tocontrol slides on which the serum would either contain or not containthe molecule of interest. This technique has application to themulti-PRE labeling and consequent detection of peptides, nucleic acidoligomers or genes, as well as portions of or whole cells or viruses.

The measurement of binding constants between two entities is currentlyperformed by several procedures. Macroscopic binding can be measureddirectly by, for example, isothermal titration calorimetry. Less directmethods include absorbance, fluorescence or changes in circulardichroism associated with complex formation. One problem associated withthese methods is that a high concentration of material is required toobserve a detectable change in signal, and at these high concentrationsthe sample may be essentially 100% complexed, thus preventing themeasurement of a binding constant under these conditions. In a preferredembodiment, the two entities are labeled with PRE probes, equilibrium isreached, and the ratio of free to bound allows calculation of a bindingconstant.

The ability to detect when two PREs are adjacent is also important forassays of molecular association and dissociation. If two PREs areassociated with suitable conjugate pairs and are mixed together, theywill bind to form a pair or, if not restricted, higher complexes. As oneexample, PREs conjugated to oligonucleotides will form such pairs orcomplexes if the oligonucleotide sequences on different populations ofPREs contain complementary sequences, or if the PRE boundoligonucleotide sequences are complementary to separate regions of atarget oligonucleotide also present in the matrix.

C. Cleavage of a Linkage Between Two PREs

In this embodiment, a PRE is linked to another PRE thorough a cleavablelinker, e.g., a peptide, oligonucleotide, oligosaccharide or otherchemically or enzymatically cleavable linker. The aim of the linkedcomposition is to detect single chemical or enzyme cleavage events, onthe basis of an observable spectral change resulting from linked PREsbecoming individual, unlinked PREs, in accordance with the Part Bembodiment.

More generally, linked pairs of PRPs, are distinguished and, if thebinding is disrupted, by, for example, enzymatic degradation of apeptide linker between the PREs or denaturation, this is reflected bythe changes in the paired or complex PRE spectra. Operation of an enzymemay be monitored by this technique by observing an increased rate ofcomplex formation or disassociation in the presence of the enzyme. Oneadvantageous application of these methods includes monitoring theoperation of a signal transduction cascade in a cell. Conjugated PREsare selected such that the presence of a product of a signaltransduction cascade either disassociates previously bound PREs or bindsdisassociated PREs. The initiation of the cascade can thus be observedwith optical microscopy in a living cell by observing association ordisassociation of PREs in the cell.

Each PRE can be coated in such a way to result in a high probability ofbound pairs by coupling with a linker such as a peptide or DNA molecule.As discussed herein, when two PREs with the same PR peak frequency arevery close to each other, frequency shifts, additional resonances andpolarization effects occur. If one wishes to determine whether aspecific enzyme is present in solution, a linker is used which issusceptible to degradation by that enzyme. For example, serine proteasescan be assayed by using a peptide linker containing a proteaserecognition site therein. After proteolysis, the spectra of the boundPREs changes dramatically as the PREs separate. In some cases, the PREsmay be spatially separated far enough apart when linked such that theydo not interact appreciably and retain essentially independentscattering spectra both when linked and unlinked. In this case, pairformation and disassociation can still be observed and measured byevaluating PRE positions with a CCD detector, and observe pairs of PREshaving relative motion which indicates that they are linked.

VI. Additional Compositions and Applications of PREs

A. Monitoring Local Dielectric Environment

When a PRE in air is surrounded with a medium having a dielectricconstant different from that of air, scattering parameters may changerelative to the scattering parameters characteristic of the particle inair. This effect is reversible if the dielectric medium is removed. Suchparameters include, for example, a change in intensity or shift inwavelength of the resonant peak, changes in the PREs response topolarized light, and a change in the number of resonant peaks. Shifts inthe resonant peak and intensity are observed following the addition ofliquids of different indices of refraction surrounding the PRE, andafter they are removed by suitable washing steps, the PREs exhibit theirprior characteristics. For many materials which exhibit plasmonresonance, raising the index of refraction of the surrounding mediumwill red shift the resonant peak to a longer wavelength.

The presence of specific substances of interest or other perturbationsin a sample being tested may therefore be detected by noting thespectral response of PREs to substances which interact with the PREs.For example, a suitable sample can be prepared having PRE bound to asubstrate. Selected molecules may be bound to the PRE surface. Theoptical scattering parameters (intensity, polarization dependence,angular dependence, wavelength dependence, etc.) of each such PRE arerecorded. The sample is then treated with material which includesmolecules of interest that selectively bind to the surface of the PRE insuch a manner that after initial treatment and/or subsequent furthertreatments, the PRE scattering parameters have changed, confirming thelocal absorption of additional material or desorption of the additionalor initial material, or other changes in the local dielectricenvironment. It can be appreciated that the initial PRE sample may beprepared as a test “library” or used to screen an “applied ” library ofproteins, antibodies, etc. These peak

(D) Shift in Fluorescence Spectrum

shifts and intensity changes can also be used to detect molecularperturbations such as association and dissociation due to changes in thePRE local dielectric environment.

Information concerning the properties of a subject matrix can also beobtained by observing the spectral dependence on the relative positionsof a PRE and a nearby substrate such as a smooth Si surface. Forexample, having made a record of a PRE location and spectral signaturein a given sample, one could add an enzyme or photolyze a bond,resulting in movement of the PRE from the substrate, thereby changingthe PRE spatial and/or spectral signature. Indeed, if a pair of such PREwere bound together, and one moved while the other remained bound to thesurface, the resulting spectral signatures would clearly indicate thisevent. Coatings on substrates can also be used to provide furtherflexibility in creating detection and analysis systems utilizing PREs.For example, a coating can be applied to a substrate which will bind adesired polypeptide or polynucleotide or a blocking coating can beapplied which will block non-specific binding of the PRE conjugates. Onesuitable coating comprises, for example, one or more layers ofdielectric materials which produce anti-reflection properties. Thecoating may also comprise one or more layers of dielectric materialwhich will produce an enhanced radiation by the PRE of the light thatenters the observing optical system. The coating can also be selected soas to displace the PRE a distance away from the basic substrate surface.A given polarization of the light scattered by the PRE will be inhibitedor enhanced depending on the distance from a reflecting surface. Forexample, if a suitable spacer layer of SiO₂ is placed on the silicon, anice point source image peaked in the center is observed as expected. Ifthe SiO₂ layer is adjusted or another dielectric substance is used,conditions can be found related to the PRE resonant wavelength and thedielectric thickness and material where there is destructive andconstructive interference of the PRE due to superposition of the lightreflected from the substrate (interface) and the top of the dielectriclayer. By using silicon or conducting surfaces, a noticeably differentspectral signature is obtained than if the PRE moves away from contactwith that surface or if a dielectric layer intervenes.

B. Monitoring Motion

Three dimensional PRE motion may be directly visualized using twoobservational lenses at right angles to each other, each yielding a twoaxis motion in the plane perpendicular to their respective optical axis.This is particularly suited to motions that are small compared to thedepth of field of the objective lens. If the motion to be observed has acomponent which is large compared to the depth of focus of the objectivelens, only one lens is used for three dimensional motion, whereby the“depth” direction is determined via a feedback signal that keeps the PREintensity in focus on the image plane. The other two dimensions aredetermined in the usual manner. PRE distance from the substrate surfacecan also be monitored in TIR illumination systems by measuring theintensity of the light scattered by the PRE as it changes position. Asthe excitation electric field drops off exponentially with distance fromthe reflecting interface, PRE intensity will decrease as it moves awayfrom the substrate surface.

Because PRE probes are so bright and so small they can be used forreal-time determination of velocity and relative motion. For example,PREs may be used to monitor dynamic cellular processes including motorproteins (i.e. kinesin), cell division, vesicle transport, etc. PREprobes are particularly useful for in vivo temporal experiments over abroad range of timescales because they do not photobleach. PREs orprecursor gold nucleating centers are attached to lipids which becomeincorporated into cell membranes. Specific PRE conjugates are designedto bind to their pair on cell surface receptors associated with the cellmembrane. This method allows monitoring of, for example, ion channelopenings. PREs may also be used to monitor movement of actin and myosinwithin muscle cells. PREs bound to or coated with conjugates can beintroduced into cells. The conjugate will then bind to its bindingpartner within the cytosol, nucleus or on various organelle membranes.Activation of cell receptors, for example, by a particular drug, canlead to morphological changes in cell structure. PREs within or on cellscan thus be used as an optical assay system for drug discovery orreceptor activation. Once bound, the PRE can be localized and its motionobserved. PREs may also be used to assay macroscopic motion. Forexample, a blood cell may be labeled and observed in circulation.Alternatively, the flow of blood or other liquid may induce acorresponding motion of the PRE. PREs can also be introduced into cellsby a Biolistic device (BioRad Inc, Richmond, Calif.) or byelectroporation.

By labeling any entity of interest with a PRE, the motion of that entitymay be monitored using the detection process described herein byincorporating a suitable real time data acquisition and analysis system.Such a system may determine motion in a three dimensional sense and, ifthe movement is confined to a plane, in a two dimensional sense. Notonly is precise information available about the motion in a specificsystem of interest, but also observable are changes in molecular motionafter drug treatment or other changes in the physical and chemicalenvironment such as alterations in temperature, pH, illumination,electric or magnetic field strength, or a change in concentration of anycompound of interest.

PREs can also be used to monitor physical motion of more macroscopicobjects. For example, a single PRE placed on an insect feeler could beused to sense its motion which could be regular or in response to anexternal molecule. This is particularly useful in detecting molecularresponses to smell and pheromones. PREs are also ideal tools forallowing analysis of mechanical motion on a microscopic orsub-microscopic scale. By binding PREs to the components of so called“nanobearings” or other micron sized machine parts, three dimensionalmotion can be visualized and analyzed on nanometer scales. In additionto the added expense of electron microscopes, motion is difficult tocapture via electron microscopy as the electron microscope is a scanningdevice, and the field of view is therefore generated over time withsequential scans, rather than viewed in real time as is possible withoptical microscopy. Furthermore, with electron microscopy, extensivesample preparation is required, in addition to an evacuated measurementarea. These factors severely limit the potential application of electronmicroscopy to real time motion analysis.

C. Near-Field Effects

The applications of PREs discussed above have focused on the far-fieldobservation of light scattered by the PREs. However, because PREs alsogenerate intense, non-radiative short-range electric fields, they may beused to affect the physical, chemical, and spectroscopic properties ofadjacent molecules in useful ways. The spectroscopic technique ofSurface Enhanced Raman Scattering may be extended to include thespecific enhancement of only those materials in the immediate vicinityof the enhancing PRE. For example, PREs may be conjugated to bind to atarget having a known Raman signature. Successful binding can bedetected by observing the surface enhanced Raman spectra of the target.They can also be useful for locally enhanced excitation and modifiedemission of nearby fluorophores. Surprisingly, PREs can produce enhancedemission from even high quantum efficiency fluorophores if the surfaceof the PRE is placed from approximately 1 to 5 nm away from thefluorophore. In contrast, it is generally thought that the presence of ametal quenches fluorophore emission of high quantum efficiencyfluorophores. This fact can be used to create fluorescent labels havinga much higher brightness or a changed lifetime, compared to when not soassociated. A label which includes a plasmon resonant conductive core(such as a silver particle of 40-100 nm diameter) and a non-conductiveshell, made, for example, from latex, may be created, wherein the shellhas fluorescent or Raman-active molecules embedded on or within it.Preferably, the peak of the plasmon resonance has a significant overlapwith the efficient excitation band for the fluorophore or Raman activemolecule. When the label is illuminated, the plasmon resonanceexcitation of the core will greatly enhance the observed fluorescence.In accordance with the above discussion, the thickness of thenon-conductive shell is preferably less than or equal to approximately 5nm in order to produce fluorescence enhancement. The plasmon resonantcore, selected to resonate at a chosen wavelength, thus dramaticallyimproves label performance over the fluorescent latex particlescurrently commercially available.

Ellipsoidal PRE responses can also be advantageously employed inconjunction with fluorescence spectroscopy. Because ellipsoidalparticles simultaneously permit resonance excitation at two or threedistinct frequencies, they are particularly effective for localizedexcitation of a selected fluorophore by one such plasmon resonance, andthen simultaneously effective for effecting the radiation (i.e.emission) of the excited fluorophore at wavelengths corresponding to theother plasmon resonance.

Thus, targeted PREs can induce very localized spectroscopic effects,again improving the collection of information about submicroscopicsystems. Similar to the case of fluorescent resonance energy transfer(FRET), clustering of PREs gives rise to new optical propertiesincluding localized and Photonic Band Gap modes, which can be used toadvantage in making highly responsive PRE-based detectors of molecularbinding events.

D. Metrology and Instrumentation

Excited PREs can produce localized heating, and an individual PRE can beused to write to a polycarbonate substrate. As individual, highlylocalized light sources, PREs can be useful in precision lithography,photochemistry, or for inducing light activated chemical reactions.

PREs can also be used as markers in conjunction with all othernon-optical forms of very high resolution microscopy, includingelectron, atomic force, and scanning tunneling microscopy. In theseapplications, a macromolecule of interest, such as a segment of DNA, ismarked with one or more optically observable PREs. Preferably, the highresolution microscope is also equipped with darkfield optical microscopyapparatus for optically observing the portions of the surface to beimaged with the non-optical microscope. The PRE's bound to the moleculecan be optically observed, and the relevant portion of the highresolution microscope, such as the atomic force or scanning tunnelingprobe tip, can be immediately positioned at a location of interest onthe molecule to be observed. This can increase the efficiency of the useof high resolution microscopes, saving excess scanning time normallyused to locate the object to be imaged. Atomic force, scanningtunneling, or any other type of scanning high resolution microscope canadvantageously be constructed to incorporate darkfield microscopysystems in order to utilize this feature of PREs.

Industrial applications requiring high precision alignment orregistration may also benefit from the use of PREs. One such applicationis the semiconductor manufacturing process, where lithographic masksmust be precisely aligned with the semiconductor wafer being processed.Because PREs can be localized to a precision of a few nanometers or evenless, a comparison of the position of one or more PREs on thesemiconductor wafer with the position of one or more PREs on thelithographic mask can determine the location of the mask relative to thewafer at the nanometer level. As only relative positioning is important,either random or controlled PRE patterns on the mask and the wafer maybe used.

Another application of the present invention takes advantage of the factthat PREs are essentially point sources of optical frequency light,having a diameter much less than the emission wavelength. Thus, theyproduce only the point-spread-function pattern characteristic of theinstrument through which they are viewed, and not an image of theirstructure. This point spread function can be analyzed to detectimperfections in the optical system used to create it. As one example,an angular variation in the intensity of the circular fringes indicatesa lens in the viewing system which has a circumferential asymmetry.Localizing the center of the Airy pattern at two or more emissionwavelengths also evaluates a lens systems for chromatic aberrations.

The point source nature of PREs can also be used to test an opticalsystem for its resolution. Using techniques described above for theplacement of individual PREs at specific locations, a calibration set ofPRE pairs can be created with varying distance between the PREs. It canthen be determined how close two PREs must be before the central peaksof their respective point spread functions overlap to produce a singlenon-differentiated peak. To some extent, the same measurement can beperformed by measuring the width of the peak of a single PRE in thefocal plane with the lens system of interest.

PREs may also be used to profile the intensity distribution of focusedlight beams, thereby gathering information concerning the properties oflenses and other optical systems used to produce such beams. Asillustrated in FIGS. 10A and B, a focusing lens 100 produces a lightbeam 102 focused to a narrow beam in the lens focal plane. As is wellknown in the art, the beam is not focused to a point at the focal plane,but the intensity has an approximately Gaussian intensity as a functionof distance away from the center of the focused beam. The details of theintensity as a function of position in the focal plane will depend onthe characteristics of the optical system which produced the focusedbeam.

Referring now to FIG. 10A, a thin transparent plate 104 may be placed inthe beam 102 at the focal plane. The transparent plate 104 includes aPRE mounted thereon. Preferably, of course, the peak of the plasmonresonance response of the PRE is selected to approximately match thepredominant frequency band of the incident light beam 102. It can beappreciated that the intensity of the light scattered by the PRE willdepend on the intensity of the illumination. Accordingly, if the plate104 or beam 102 is moved such that the PRE moves to different locationsin the focal plane, the intensity as a function of position can bedetermined by collecting scattered light with a suitably placedobjective lens of an observing microscope. As with other darkfieldtechniques described above, the objective of the observing microscopemay be placed so that it collects light emitted by the PRE but does notcollect light transmitted through or specularly reflected by the plate104. This system may be used to test the characteristics ofsolid-immersion-lenses, lasers, and other optical systems.

E. Object Identification

Still another application of the present invention is the labeling andidentification of paper or plastic items subject to forgery such aspaper currency or credit cards, or identification badges. Either randomor pre-defined patterns of PREs may be bonded to the surface of theitem. In advantageous embodiments, the PREs are coated with a protectivelayer or film. Later observation of the proper PRE pattern on the itemwith microscopy techniques as described above can be used forauthentication purposes. Such authentication can be implemented via apattern recognition system on a computer, allowing for real timeauthentication at point-of-sale terminals, facility entry locations, andthe like. Alternatively, a magnetic strip, bar code, or other datastorage media may be placed on the item (e.g., a credit card) inaddition to the PRE arrangement. A coded version of the PRE array isalso stored in this media, and a match indicates that the item wasvalidly created. Of course, a cryptographic algorithm which produces amatching magnetic code based on the PRE array that cannot feasibly bededuced from the array itself should be used, and such algorithms arewell known in the cryptographic art.

G. Forensics

The robustness and easy visibility of PREs also makes them ideal forseveral forensic applications. Bodily oils, fluids, DNA, etc. which ispresent in fingerprints can bind PREs, making the fingerprint easilyvisible under appropriate illumination. Many different goods may also belabeled with PREs to provide traceability. PREs having differentscattering characteristics can be mixed with explosives, food, drugs,poisons or other toxins, etc. The particular PRE could provide sourceidentification. PREs are ideal for this application because of theirresistance to degradation and the ability to detect even singleindividual PREs in a sample.

H. Identifying Small Molecules in Combinatorial Libraries by RamanSpectrum PREs

PREs can also be differentiated by the characteristics of moleculeswhich are attached to their surface which may be provided in addition tothe one or more conjugate molecules. Surface enhanced Raman scatteringfrom Raman active molecules adjacent to individual PREs has beenreported (Nie and Emory, Science, Volume 275, 1102-1106, 1997). Ifmolecules with different Raman spectra are attached to differentpopulations of PREs, PREs from different populations may be identifiedby their different Raman scattering signatures. Given the wide varietyof Raman molecules available, a large number of differentiable probesare possible which may be particularly useful in conjunction withcombinatorial library techniques. The use of Raman markers may also beused as an alternative way (in addition to four different plasmonresonance wavelengths) to produce four differentiable PRE populations,which would be useful in DNA sequencing techniques which use fourdifferentiable markers, one for each base. Fluorescent molecules mayalso be bound to PREs to provide an additional marker, as canoligonucleotides, which are distinguished by their preferentialhybridization properties rather than spectrally. If desired, PREs havinga conductive resonant core and a non-conductive dielectric shell such aslatex may include embedded fluorescent molecules in the dielectricshell. This label embodiment is discussed in more detail below. It canbe appreciated as well that combinations of different resonantscattering characteristics, different fluorescent markers, and differentRaman markers can be utilized to prepare hundreds or even thousands ofspectrally differentiable probes. For example, a library may includefour different plasmon resonance signatures, four different fluorescentsignatures, and ten different Raman signatures, thereby producing 160different distinguishable probes by different combinations of theavailable spectral signatures. Accordingly, populations of PREs may bedistinguished based on differences in size or shape, or by differencesin material bound thereto.

Furthermore, known techniques of combinatorial chemistry can be used tosimultaneously synthesize a marker molecule and a conjugate moleculeonto PREs in a simultaneous series of molecular assembly steps. In someembodiments, this process would start with a label precursor entitywhich comprises a PRE having one or more reactive groups bonded to itwhich may form a base on which combinatorial chemical synthesis mayinitiate. The reactive groups may include, for example, a phosphates,aldehydes, carboxyls, alcohols, amides, sulfides, amino acids, ornucleic acid bases. For example, a selected Raman active molecule couldbe synthesized simultaneously with an oligonucleotide conjugate.Alternatively, a library of drug candidate compounds may be synthesizedsimultaneously with identifying oligonucleotide markers.

I. Cell Sorting

PRE probes are also suitable for cell sorting, analogous to fluorescentactivated cell sorting (FACS). A mixed cell population is analyzed forone cell type expressing a particular surface antigen using a particularPRE probe. In addition, several cell types are isolated bysimultaneously using multiple PRE probes because of the number ofuniquely identifiable PRE probes with distinct spectral signatures thatcan be made. It is contemplated that all of the PRE detection andlocalization methods described herein can be fully automated to produce,among other items, cell sorters. With a PRE cell sorter, it isadvantageous to pass the cell population of interest substantially oneat a time into the field of view of a darkfield microscope. Thedetection, discrimination, and analysis techniques described in detailabove can be used in the cell sorting context to identify PRE labelledcells.

Many different cell routing schemes may be used in such an apparatus. Inone advantageous embodiment, the cells are deposited into a stream offluid, such as water, which is constrained to move within the confinesof a surrounding shell of a second fluid, such as an oil, which issubstantially immiscible in the first fluid. This forces the cells toremain confined to a small region for darkfield viewing as they passthrough the field of view of the microscope. Preferably, the indices ofrefraction of the two fluids are approximately equal, to minimizereflections of incident light at the interface between them.

In addition, PRE labeling can be used in addition to, rather than as asubstitute for, fluorescent labeling in a cell sorting technique. Inthis case, fluorescent labels and PRE labels are made to bind to thesame target cells. The cell sorting may be done based on an observationof the fluorescent marker. If a portion of the sorted cells are saved asan archival record of the result of the sorting process, the PRE can beused to verify successful sorting in the future. This is more effectivethan observing the fluorescence of stored samples, due to the stabilityand non-photobleaching properties of the PRE.

A further application of the same technology is performed in vivo or exvivo. In this technique, cells are permeabilized and PRE probe(s)attached to antibodies against a cellular biomolecule of interest areintroduced into the permeabilized cells. The cells are then incubatedwith a combinatorial chemical library. The viable cells are spread outon a slide and the cells are selected which have been “affected” by thechemical library. “Affected” could indicate a change in localization ordistribution of PRE probes due to a change in localization of theattached biomolecule, or it could indicate a clustering of PRE probesleading to a new spectral signature. Because any entity of interest(i.e. cell, DNA, organelle) can be labeled with a PRE, it can then beoptically detected because the collection of PRE can be observed movingas a unit.

J. Clinical Applications of PREs

PREs can also be used in a wide variety of clinical applications. Onesignificant area is in the diagnosis of different conditions in animals,including humans, which can be identified by the selective binding ofconjugate to specific organs in the animal. In this technique, PREshaving selected scattering characteristics may be injected into thebloodstream or ingested by the animal. These PREs may further be boundto an antibody or other conjugate to target or identify the presence ofa particular substance in the animal. Tissue may then be removed formthe animal and tested for the presence of PREs under a microscope. Ifdesired, control PREs which are not bound to the specific bindingconjugate can also be injected or ingested to determine the non-specificbinding background. These techniques have been developed with coloredlatex particles as the probe, and reagents for performing these testswith the latex particles are commercially available from, for example,Triton Technologies of San Diego, Calif. and Molecular Probes of Eugene,OR. The use of PREs, due to their brightness, biocompatibility, andresistance to degradation will improve the sensitivity of such tests.

Cell modification and therapy techniques such as gene therapy may alsobe enhanced with PREs. In this case, cells having the desired geneticcharacteristics are labeled with PREs and selected with a cell sorterusing the techniques set forth above. Selected cells are then placed ina patient. If desired, the PRE can be disassociated and removed prior toplacement in the patient.

Selective heating and drug delivery is also possible with PREs. If PREsare localized in a selected tissue or region of a patient, they can beilluminated so as to locally heat the tissue or region withoutsignificant affect on neighboring areas of the body. The administrationand activation of light activated drugs is also enhanced with PREs.Light activated drugs can be activated with far less total light energyby being bound to a PRE where the electric field will be enhanced. Theuse of light activated drugs to treat breast cancer has received recentattention, and may be improved by binding the drugs to PREs to enhancetheir activation at locations deeper in the tissue.

The application of optical PRE detection and analysis to biochemicalsystems is considered to provide many advantages over current labelingtechniques, and appears to comprise an area where PRE analysis can havea large impact. Other areas, however, may also benefit from the PREdetection and spectral analysis of the present invention.

From the foregoing, it will be appreciated how various objects andfeatures of the invention have been met. The method and apparatus of theinvention are ideally suited to a variety of target-interrogation tasksthat have been difficult or impossible heretofore, including, asrepresentative examples:

1. detecting single molecule events;

2. resolving sub-wavelength distance relationships in a biologicaltarget in a natural hydrated state;

3. direct spatial mapping of selected target sites on a biologicaltarget, such as direct mapping of selected sequences in a chromosome forpurposes of chromosome mapping; and

4. optical reading of microencoded information;

The method and apparatus can further be applied to a wide variety ofdiagnostics applications, to achieve improved sensitivity, spatial anddistance information, ease of sample preparation, and flexibility in thetype of target sample that can be interrogated.

Although the present invention has been described with respect toparticular methods, compositions, and devices. It will be appreciatedthat various changes and modifications can b made without departing fromthe invention.

What is claimed is:
 1. A method of interrogating a field having aplurality of plasmon or resonant entities (PREs) distributed therein,comprising; illuminating the field with an optical light source,detecting a spectral emission characteristic of individual PREs andother light scattering entities in the field, constructing a computerimage of the positions and values of the emission spectralcharacteristic of individual PREs and other light-scattering entitiespresent in the field, and discriminating PREs with a selected spectralsignature from other light-scattering entities based on detectedspectral characteristic values unique to the selected-signature PREs, toprovide information about the field.
 2. The method of claim 1, whereinsaid detecting includes simultaneously detecting the spectral emissioncharacteristic of the light-scattering entities in the field.
 3. Themethod of claim 2, wherein said detecting further includes detecting thespectral emission characteristic of the light scattering entities in thefield simultaneously at a plurality of defined spectral frequencies. 4.The method of claim 1, wherein said illuminating and detecting stepsinclude: illuminating said PREs with incident light predominantly in afirst frequency band; detecting the spectral emission characteristics ofindividual PREs and other light scattering entities in the field underillumination at the first frequency band; illuminating said PREs withincident light predominantly in a second frequency band; and detectingthe spectral emission characteristics of individual PREs and other lightscattering entities in the field under illumination at the secondfrequency band.
 5. The method of claim 1, wherein said detectingincludes sequentially detecting the spectral emission characteristic ofindividual PREs and other light scattering entities in the field at aplurality of defined spectral bands.
 6. The method of claim 1, whereinsaid illuminating includes exposing the field to a plurality ofnarrowband pulses of light which vary in duration, and said detectingincludes detecting variations in emitted light intensity produced byvariations in duration.
 7. The method of claim 1, wherein at least someof the PREs are non spherical, said illuminating includes exposing thefield to polarized light at different orientations and/or differentangles of incident, and said discriminating includes detecting changesin a spectral emission characteristic as a function of incident lightpolarization orientation or angle.
 8. The method of claim 1, whereinsaid PREs are formed in the field by a step selected from the groupconsisting of: (i) binding nucleation centers to a field, metalenhancing said nucleation centers, observing enhancement of saidnucleation center during said metal enhancing process, and terminatingenhancement when a PRE of a desired spectral characteristic has beenformed; (ii) adding pre-formed PREs to a target in the field, (iii)making PREs at target sites in the field.
 9. The method of claim 1,wherein discriminating PREs with a selected spectral signature fromother light-scattering entities in the field includes discriminating aselected type of PRE from all other light-scattering entities in thefield, based on detected values, for each light-scattering entity in thefield, of peak position, peak intensity, or peak width at half intensityof the spectral emission curve, peak halfwidth in the image plane, andpolarization or angle of incidence response.
 10. The method of claim 9,wherein said discriminating is effective to discriminate, for a selectedtype of PREs, those selected PREs which are interacting with one anotherand those which are not.
 11. The method of claim 9, wherein saiddiscriminating is effective to discriminate a selected type of PRE fromanother selected type of PRE in the field.
 12. The method of claim 1,wherein the PREs have surface-localized fluorescent molecules orRaman-active molecular entities, and said detecting includes detectingplasmon-resonance induced fluorescent emission or Raman spectroscopyemission from one or more of said molecules or entities, respectively.13. The method of claim 1, for use in determining the total number ofPREs of a selected type in a field, wherein said discriminating includescounting the number of PREs having a selected range of values of aselected spectral emission characteristic in the constructed computerimage.
 14. The method of claim 1, for use in determining a spatialpattern of PREs having a selected range of values of a selected spectralcharacteristic in the field, wherein discriminating includesconstructing an image of the relative locations of PREs with thosespectral-characteristic values.
 15. The method of claim 14, wherein thelocation between two adjacent PREs is less than the Rayleigh resolutiondistance, and said detecting includes exposing the field with light ofone wavelength, to obtain a diffraction image of PREs in the field,exposing the field with light of a second wavelength to obtain a seconddiffraction image of PREs in the field, and comparing the distancebetween peaks in the two diffraction patterns.
 16. The method of claim1, for use in interrogating a change in the environment of the field,wherein said discriminating includes comparing the values of thedetected spectral characteristic of a PRE in the field before and aftersaid change.
 17. The method of claim 16, wherein the field isinterrogated for changes in the dielectric constant of environment. 18.The method of claim 1, for use in detecting motion of PREs in the field,wherein said illuminating is effective to generate, for each PRE, adiffraction pattern having a center peak and said detecting includesdetecting the center peaks of the diffraction patterns of the PRE's inthe image plane, as a function of time.